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E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.
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a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 µm. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTPneonates.
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(F) The proposed working model. In brief, we uncover that sugar metabolism is rewired to protect genome stability by means of a redox mechanism. Mechanistically, an SNF1-Mig1-ZWF1-NADPH axis stimulates the ssDNA-binding of RPA, thereby promoting a fast activation mode of Mec1-Rad53 kinase cascade to fight against replication threats.
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(top) Western blot analysis for iNOS and tubulin (n=3). (bottom) Media nitrite levels (n=3).
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A) Acetylation tracks for H3K9/14ac and H3K27ac ChIP-seq (on the left) and corresponding FPKM values by RNA-seq (on the right) for representative genes in YC, YT, OC and OT FAPs.
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(O) Wound area measurements in Col1α1-TK and WT littermate mice. WT, N=13; Col1α1-TK, N=12 biological replicates, data are from two independent experiments. Two-way ANOVA performed comparing WT to TK mice.
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Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice. A. Western blot shows enhanced eIF2 alpha phosphorylation (top) and RT-PCR demonstrates increased XBP1 mRNA splicing (bottom) in denervated muscle. Right panels show relative quantification of signal intensity. *p<0.05 by Student's t test.
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Fluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 μg/ml of aCD3/aCD28 in the presence of human 10 μg/ml of PD-L1 protein and 100 nM of MB for 6 hours. Luciferase activity was measured as readout for CD28-P85 interaction Data information: Data are representative of three independent experiments, and were analyzed by unpaired t-test. Error bars denote s.e.m. *P < 0.05; **P < 0.01; ****P < 0.0001
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NoV levels in the vaccinated mice hindlimb muscle tissue at 2, 3 or 4 days post infection (dpi) with WT NoV were determined by Western blotting to detect the viral coat protein (CP) and B2 Staining of β-actin was used as a loading control (B).
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(D) Quantification and representative images of CA9+ tumor hypoxic areas in vehicle and glufosinate (20mg/kg) treated mice (n=6). Six images per tumor were analysed. Scale bar: 50µm.
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(A) Representative images from 6 h infected WT BMDMs with JR32 or legA9 mutant bacteria. Nuclei were stained blue with DAPI and L. pneumophila stained green with L. pneumophila antibody. Lyso−tracker red was used to stain acidified lysosomes. White arrows show L. pneumophila colocalization with lysotracker.
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(F-I) Terminal cells expressing the general membrane marker CD4::mIFP in combination with different membrane or polarity markers: PH::mCherry, a PIP2 sensor commonly used as apical plasma membrane marker (F'), an endogenously GFP-tagged Crb (G'), polarity proteins Par3::YFP (H') and Par6::mCherry (H''). The boxed region in (H''') is shown at higher magnification in (I) over 4 time points. Red arrowheads: CD4::mIFP vesicles and associated markers at the tip of the cell.
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I. U2OS cell lines conditionally expressing indicated GFP-FAM111A alleles were fixed at the indicated times after treatment with Doxycycline (DOX) to induce expression of the transgenes and stained with crystal violet.
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(J) Glucose tolerance test results following intraperitoneal glucose challenge in Abca12tm1d and cre control mice fed normal chow or HCD (mean±SEM, n=3 mice per genotype, *p=<0.05, for tm1d versus tm1d+HCD, Students t-test).
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TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1-CFP, ANO8-YFP and Orai1-mCherry before and after store depletion. (A) Example images. (B) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone (black) or together with ANO8 (red). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.
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F qRT-PCR analysis of expression of the indicated SLC family transporters in MeWo (left) and A375 (right) melanoma cells after treatment with LDHAi for 48 hr. Data information: Statistical analysis was performed by ordinary one-way Anova for the comparison of more than two groups. Unpaired t-test was used for the comparison of two groups N.D., not determined. Data are shown as the mean ± SD, n = 3. ns: not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
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A, B ChIP-qPCR analysis of Scc1 association with early replication origin ARS305 or ARS607. HA-tagged Scc1 interaction with chromatin was monitored in wild type (Y39) and scc2-4 (Y4366) cells. Error bars represent mean value ± standard deviations of mean (n=3)
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Western blot analysis to determine levels of nucleic acid sensor proteins with lysates of (C) HT29 cells stably expressing control shRNA or IRGM shRNA,
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Detection of endogenous Rabl2 in purified multicilia. Multicilia purified from cultured mEPCs
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(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).
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(A, B): ER Ca2+ content was measured with ER-GECO1 in the periphery (A) and the center (B) or cells transfected with M3 receptors and with (red, green) or without (black, blue) ANO8. Receptor mediated store depletion was initiated by stimulating the cells with 0.5 mM carbachol in Ca2+-free solution. Cell stimulation was terminated with 10 µM atropine and ER Ca2+ uptake was initiated by perfusing the cells with a solution containing 5 mM Ca2+. The fluorescence was measured as F/F0 and normalized to the initial fluorescence in the presence of ANO8. Panel (C) shows traces of ER Ca2+ uptake at expanded time scale in the periods marked by rectangular in (A and B) and the averaged slopes of ER Ca2+ influx and (D) shows the averaged slope of Ca2+ release at the cell periphery (Peri) and cell center (Center). In (C) the traces were aligned along the Y axis to better show the difference in uptake rate. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.
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Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). SM represents Birinapant.
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] |
b, Mouse liver LC3 immunogold. Insets show higher magnification. Arrowheads indicate gold particles (black) and LC3-labelled bilayer membranes (white). c, Percentages of autophagic vacuoles (AVs) containing only lipid (Lipids, *), other cargo (Other) or mixed cargo (Mixed) in cells treated as in b (*P 0.01, **P 0.001 with cells in RM, n = 46). Left: representative examples. Error bars, s.e.m.
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G. Using high power, high resolution laser scanning confocal microscopy, no human LAG3 signal could be detected in human neurons (Auto-hLAG3 transduced, DOX OFF) by two different anti-human LAG3 antibodies (17B4 and D2G40; left panel and zoomed-in insets) whereas LAG3 was clearly detected in human neurons induced to express hLAG3 (DOX ON; right panel and zoomed-in insets). Scale bars 25 µm.
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(D) Confocal microscopy of THP-1 cells treated sequentially with LPS and LLOMe, and stained for LC3B and TRIM16. Line tracings correspond to arrows. Arrowheads indicate colocalization. Scale bars, 5 μm.
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(C) GST pulldown of recombinant GST-Ythdf or GST alone mixed with recombinant 6xHis-NT-Fmr1. 2 µM of recombinant proteins were using during the pulldown. The pulldown of the proteins was analyzed by CBB staining.
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Drp1WT and Drp1S616D, but not Drp1S616A, rescue mitochondrial structures in PINK1KO flies. Representative images of Mito-GFP (green) labeled mitochondria (d) from PINK1WT flies expressing mhc-gal4 (Mhc/+) (as a control) and PINK1KO flies expressing either mhc-gal4 (Mhc/+), Mhc-gal4 driven human Drp1wt (Mhc>hDrp1wt) or Mhc-gal4 driven human Drp1S616A (Mhc>hDrp1S616A) or mhc-gal4 driven human Drp1S616D (Mhc>hDrp1S616D) are shown. Scale bar=10 μm (d) Average mitochondrial size was quantified, >3 flies/group and 3-6 pictures of different microscopic fields from each fly were analyzed per repeat. One-way ANOVA followed with Tukey's test. *p<0.05, ***p<0.001. Data was presented as mean ± SEM of three independent experiments (e).
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(C) Heatmaps of the standardized expression of the GO iron ion import pathway genes in HIV-1 infected and mock infected samples over time. Expression levels were standardized [(mean gene expression - SD)/SD] for each gene in each time point. The TFRC (TfR1) gene, which was considered for further analysis, is highlighted.
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(C) Serine production from glucose (serine m+3, 13C6-glucose labelling, left panel) and glutamine (serine m+1, 15N2-glutamine labelling, right panel) in Flp-In 293 HA-GFP or Flp-In 293 HA-IKKε cells treated with doxycycline (50 ng/ml, 16 hours) metabolite levels were normalised to the internal standard HEPES.
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Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).
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Anti-hTAPBPL mAb (clone 54) neutralized the inhibitory activity of hTAPBPL-Ig on T cell proliferation Significance in was calculated by one‐way ANOVA with Dunnett test and others by two‐tailed Student's t‐test. * P<0.05, anti-hTAPBPL Ab group was compared with isotype Ab group.
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Schematics of EdU incorporation assay. Drugs were applied to RPE1-hTERT cells 12h after initial cell plating. 6 days later (144h), cells were switched to drug medium containing 5-ethynyl-2'-deoxyuridine (EdU; 10 μM) for 24 hours before fixation and analysis.
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B. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to ectoderm. C. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zfp354c-/- mESCs in the 3KI background to mesendoderm. D. Differentiation of Atf1-/-, Fos-/-Jun-/-, Foxj2-/-, Meis3-/-, Nr5a2-/-, Sp1+/- and Zff354c-/- mESCs in the 3KI background to definitive endoderm.
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A scheme of parametric decomposition. (i) Start from protein abundance measurements in different timepoints following stress. (ii) Fit RNA parameters based on a simplified model for transient transcriptional response (Weiner et al, 2012) (iii) Compare different conditions/perturbations in parameter space.
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D. Muscle fiber size (100 fibers/mouse) was quantified from wild type (black, n = 5), CHOP −/− (white, n = 3) and Beclin-1 +/− (grey, n = 3) mice 7 days post sciatic nerve transection. Shown is relative fiber cross sectional area (mean +/− SEM). ***p<0.001 by ANOVA.
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C, D Double-plotted actograms of NHPs' activity for 16 days. NHPs were treated with 10 mpk PF-670 for 3 consecutive days at the same solar time of day: 4 pm for the DD experiment (C) and 4:30 pm (ZT11) for the LD experiment (D), respectively, which are highlighted as red lines. White and black rectangles indicate the times of light going on and off (LD 12:12).
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C. Plot comparing chromosome-wide association of Rif1 and Rif1-∆C594 with R loop-prone sites. Plot shows ChIP profiles of Rif1 and Rif1-∆C594 across entire chromosome VI. R loop-prone sites are marked in green ('DNA:RNA hybrid' track in green), as previously assessed by S1-DRIP-Seq analysis. Also shown are positions of predicted G4-forming sequences ("Predicted G4" track in orange), and positions of replication origins (ARS) and centromere (CEN6)
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IFN-β protein (K) levels in WT BMMs treated with EVs for 4 and 24 hr respectively. The EVs were isolated from BMMs that were infected with the different M.tb Erdman strains. Data shown in K) are the mean ± SD (n = 3 per group) and all data shown are representative of three independent experiments.
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A. Parental HEK293T, HET, HOM1 and KO1 cells were treated with the indicated ligands for 1 h. Data information: In all cases, whole cell extracts were Western blotted using the indicated antibodies. Representative experiments are shown. The levels of pSMAD1/5 relative to Actin were determined and are expressed as a fold change relative to the parental HEK293T untreated sample Act, Activin A; Par, parental HEK293T cells, KO1, ACVR1 KO clone 1.
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A. The relative mRNA levels of BZLF1 and BRLF1 at 24 hpi following EBV infection of HK1 cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV-GFP and analyzed by qRT-PCR at 24 hpi. All the gene expression levels were normalized to the housekeeping gene ACTB.
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F Liver sections from indicated mice were stained with anti-KDEL antibody. Arrowheads indicate ER stressed hepatocytes. Scale bar 25 µm. Data information: con (n=3) mice were fed with conventional sterilized food, -q (n=3) mice were fed with a q-free synthetic diet for 60 days, and +q (n=3) mice were fed with the same synthetic diet supplemented with 40 nM queuine for 60 days.
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C. GO term analysis of significantly enriched genes (FDR<0.2) in CRISPR-Cas9 screen in (A), using Reactome pathways from PANTHER16.0.
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(C) Heatmap showing the MS spectral counts of high confidence interactors (FDR < 0.05 after SAINT analysis) identified with WT AR-BirA* and AR W752R-BirA* upon DHT or vehicle exposure. Values for three biological replicates from DHT-and vehicle-exposed samples are shown. GRPEL1 and DBT were the only high confidence interactors of AR W752R-BirA*. On the right, FDRs and spectral count averages are shown for the DHT-treated samples. Spectral counts have been normalized to those of AR in each sample.
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(D) Trypan blue cell death staining of Arabidopsis seedlings challenged with Pst DC3000 or Pst DC3000 AvrRpm1 (AvrRpm1) for 48 h.
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C. mRNA levels of indicated RP transcripts determined by qRTPCR in MEFs transfected with NTC siRNA (white) or p300 siRNA (grey). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
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B) RNAscope in situ hybridisation analysis of TEAD1/2/4 mRNA expression from control (Cre negative) Lats1flox/flox Lats2flox/flox animals and Villin-CreERt Lats1flox/flox Lats2flox/flox animals treated with tamoxifen to induce homozygous deletion of Lats1/2 (dKO). Tamoxifen treated (3 days i.p.) Villin-CreERt Lats1flox/flox Lats2flox/flox double homozygous mouse intestines show elevated expression of TEAD1 and TEAD4 in both the small intestine (top) and colon (bottom). Tissues were harvested and analyzed at day 7 after first tamoxifen injection. (n=5 animals for each genotype)
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(C, D) Immunoprecipitation of Bcl‐2 and Beclin‐1 in subcellular fractions. Cells treated as in (B) were lysed and subjected to subcellular fractionation to enrich either ER vesicles (microsomes, (C)) or mitochondria (heavy membrane fraction, (D)), followed by immunoprecipitation of Bcl‐2 and immunodetection of Beclin‐1. Calreticulin and Hsp60 were used as internal controls. Results are representative of three independent determinations.
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Cell viability upon 72 h combinatorial treatment with 0-20 µM cisplatin and 25 µM LJH685 in patient-derived CAFs
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(A) MCF10A cells were lysed and anti-TRIP6 or control (IgG) antibodies were used to isolate immune complexes. Immune complexes and lysates were probed by western blotting for vinculin and TRIP6.
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C: Dose response curves of rhabdoid tumor and Ewing sarcoma cell lines. RT cell lines (black) are sensitive to mithramycin treatment with a similar IC50 value as TC32 ES cells (grey). RT cell lines are not sensitive to three broadly-active chemotherapeutic agents: etoposide, doxorubicin or SN38. Data represents mean with standard deviation derived from three independent experiments.
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(D) Frequency of apnea of 9 WT, 10 KO, 14 KO/HTTSD, and 9 KO/HTTSA mice at P35 and P55. (Kruskal-Wallis test with Dunn's comparison). Data information: Data are presented as the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant.
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(B) Quantification of the number of mCherry/GFP double‐positive and mCherry single‐positive puncta per cell in (A). Each bar represents the average and s.e.m. of 30 cells per condition in three independent experiments. The number of mCherry single‐positive puncta of Ubqln1 and Ubqln4 siRNA was compared with that of control siRNA. *P‐value=0.0475 and **P‐value=0.0373. GFP, green fluorescent protein; LC3, microtubule‐associated protein light chain 3; Ubqln, Ubiquilin.
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Cell viability assays were performed as described in Materials and Methods after cells were treated with temozolomide (A), cisplatin (B), adriamycin (C), taxol (D), TRAIL (E), bafilomycin A1 (F), thapsigargin (G), and MG132 (H). Means ±S.D. from at least three independent experiments are shown as relative indexes after normalization to those of individual control cells treated with DMSO.
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Histologic assessment of the number of TH+ mDA neurons in the SN and TH+ fiber intensities in the striatum Shown are the representative images for TH+ DA neurons in the TH-immunoreactivity in the striatum,
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G. HA-ubiquitin and Flag-FoxM1 were co-transfected with or without GSK3β CA into 293T cells. After 36 hr, cells were treated with 25 nM MG132 for 6 hr. Cell lysates were subjected to IP with Flag followed by IB with HA or FoxM1 antibody.
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(C) LPS/ATP treated and non treated wild type and Becn1+/- microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non stimulated cells ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1+/- microglia; mean ± SEM, wild type LPS/ATP n = 4, Becn1+/- LPS/ATP n = 7; two-tailed t-test *: p < 0.05; scale bar: 50 µm, insert scale bar: 10 µm.
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Schematic diagram of the position of two phosphorylation sites on the BCL9 N-terminal domain, and protein alignment analysis of the sequence around T172 in different species from http://www.uniprot.org.
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5-week-old Sel1Lf/f and Sel1LAlb mice were injected i.v. once with AAV8-shCrebh or control AAV8-shLuc. Insulin tolerance test (ITT) 5-weeks after injection (n=10 per group).
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C. Position of Cα atoms along MD simulations of apo-form (red) and the pre-stem hairpin extraction form (blue) are shown with the lectin modules superimposed (200 structures for each).
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Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel.
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C,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.
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(E-F) Confocal images of sections proximal to the injection site in Q2 (E) and distal to the injection site in Q4 (F), showing Cnga3 expression in PNA-positive cone outer segments. OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.
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(F) NanoSight analysis of EVs in CSF of control (black) and TNF-injected (25 µg/20 g; grey) mice 6 hours after TNF challenge.
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(B) mcp3 cells have normal mitochondrial morphology. WT and mcp3∆ cells expressing mitochondrially targeted GFP (mtGFP) were grown to mid-logarithmic phase then analysed by fluorescence microscopy. Typical images of the two different strains are shown (scale bar = 5 µm). The quantification of the depicted strains shows the average percentage with standard deviation bars of three independent experiments with at least 100 cells per experiment (n=3; SD; p as indicated).
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Distribution of rotation frequencies in trapped sperm (n = 32; mean frequency ± SD 6.0 ± 2.1 Hz).
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(B) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows.
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(a-d) RNAi-mediated knockdown of dUTX inhibits ecdysone-induced apoptotic cell death. SL2 cells were treated with two independent dsRNAs to dUTX or a control gene (GFP) for 48 h and cell death was induced with 10 µM ecdysone for 24 h. (c) Caspase activity was measured from total cell lysate on DEVD-AMC, represented as fold increase relative to control RNAi. Data are mean from three independent experiments, with error bars representing s.e.m. *P0.05 (Student's t-test).
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Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change).
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C) Model for the role of TBC1D14 and the TRAPP complex in mammalian autophagy and ATG9 traffic. Recycling endosomes harbour a population TBC1D14 bound to RAB11, and a population of ATG9 molecules which may traffic to and from the ATG9 compartment. (i) Upon amino acid starvation, TBC1D14 and RAB11 induce a vesicle trafficking step from RAB11 to RAB1 positive membranes at a tubulo-vesicular transport intermediate (ii). This results in recycling of ATG9 to RAB1-regulated Golgi compartments (iii) from where ATG9 can be trafficked to the ATG9 compartment in an ULK1 dependent manner (iv). This maintains the ATG9 compartment under starvation, which can then both contribute to, and promote autophagosome formation from other membrane sources, including the RAB11 positive recycling endosome (v).
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i, representative flow cytometry analysis of Nrl.Cre+/- & mTmGfloxed co-cultures. Samples were analyzed for expression of myrGFP (recombination) and CD73 (photoreceptor identity) versus CD73-ve fraction (other retinal cells). N > 3 independent cultures for each condition with technical triplicate for each sample; One way ANOVA, non-parametric, Kruskal-Wallis with Dunns' multiple comparisons test
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I. MitoTracker Green staining. p-value was calculated using two-sample t-test. The numbers for each genotype are mean fluorescence intensity.
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Time-of-addition study with selected FDA-approved drugs.Five FDA-approved drugs were analyzed by time-course experiments to determine the stage in the MERS-CoV life cycle that is inhibited by the drugs. Vero cells were infected at a multiplicity of infection (MOI) of 5 with MERS-CoV, and FDA-approved drugs were administered at six-time points prior to or post-infection as indicated. Drug concentrations were higher than the 90% inhibitory concentration (IC90) values of the drugs, and chloroquine was used as an early stage infection inhibitor control.
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(C) Generation of NDST3 polyclonal antibodies targeting a region of NDST3 unique in the NDST family.
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mice body weight (C) in vehicle and glufosinate (10mg/kg) treated mice (pool of 2 independent experiments; 10 mice per condition in total).
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Length distribution of CldU tracks 45 minutes after release into S phase in the DNA combing experiment shown in (D). Median values are indicated in red. For each sample, at least 250 tracks were analyzed in two biological replicates.
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(c) Aggregation in COS-7 cells transfected with either pcDNA3:1 (empty vector) or constitutively active (CA) m-calpain and EGFP-HDQ74 (3:1 ratio) for 4 h and then treated with or without 500 μM 2′5′ddA for 48 h. Error bars show 95% confidence interval.
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G: Pds1 signal intensities from western blots shown in F were quantified and corrected for Pgk1 signal of the corresponding sample. Finally, the highest signal intensity (45 minutes after release) was defined as 1.00 and values for other time points were normalized accordingly.
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C, Higher-energy collisional dissociation (HCD) spectrum of TFEB-C212 containing peptide (VGVTSSSCPADL), left top panel indicates the assigned b- and y- fragment ions, and left bottom panel their mass deviations from corresponding theoretical values showing glutathionylation at Cysteine 212. Right panel shows extracted ion chromatograms (XIC) of TFEB glutathionylated peptide from cells either untreated (Control) or treated with NaAsO2.
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(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.
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E. Histochemistry of respiratory enzymes, COX, NADH, and SDH on testical sections from mice 10 days postnatal. Scale bar: 100 m.
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(g) Box-whisker plots of the numbers of circulating endothelial cells per million PBMCs in healthy (n = 6) and NAFLD subjects (n = 11). Box-whisker plots indicate median (middle line), 25th, 75th percentile (box) and the lowest/ highest data points (whiskers). ns, non-significant (t-test).
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A Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue) on frozen sections of limbmuscles injected with myoblast clones expressing different VEGF levels (V Low, V Med, and V High, respectively) and treated with VEGF-Trap or saline after 10 and 17 days. Analysis was performed after 4 days of treatment (2 and 3 weeks, respectively). All normal vessels induced by low and medium VEGF doses displayed a similar coverage by normal pericytes, whereas aberrant structures induced by high VEGF were covered with α-SMA-positive smooth muscle cells. Asterisks indicate muscle fibers, which were mostly sectioned in longitudinal orientation, although some cross sections are visible depending on the orientation of muscle bundles. Scale bar = 25 μm.B, C Quantification of vessel length density (VLD) on the same samples showed that stabilization started already by 10 days for vessels induced by low VEGF, but not until 17 days for those induced by medium VEGF, despite similar pericyte coverage, while aberrant structures induced by high VEGF always regressed. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group; 2 weeks (B): V Low saline, n = 9; V Med saline, n = 13; V High saline, n = 8; V Low TRAP,n = 28; V Med TRAP,n = 22; V High TRAP,n = 30; 3 weeks (C): V Low saline, n = 9; V Med saline, n = 11; V High saline, n = 19; V Low TRAP,n = 43; V Med TRAP,n = 14; V High TRAP,n = 19; *P < 0.05 and ***P < 0.001 (all versus Ctrl) by one-way ANOVA with Bonferroni multiple comparisons test; 2 weeks (B): V Low TRAP versus Ctrl P < 0.0001; 3 weeks (C): V Low TRAP versus Ctrl P < 0.0001; V Med TRAP versus Ctrl P = 0.0158.
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(C) Sulfo-NHS-biotin was administered via intraperitoneal injection 10 min before perfusion for assessing the blood-brain barrier permeability, i.e., extravasation is indicative of leakage from the intravascular lumen. Sulfo-NHS-Biotin was confined to the capillaries of the meninges and cortex (as shown with arrows), and there was no evidence of extravasation. Scale bar = 100 μm.
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D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.
|
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H, MiaPaCa-2 cells were transfected with empty vector (e.v., control) or Flag-tagged wild type (wt) p14ARF-containing plasmid and treated (+) with 20 μM MS023 or left untreated (-) for 3 days. For the last 2 days, the cells were additionally exposed to 1 μM gemcitabine (+ GEM) or not (- GEM). p14ARF overexpression was monitored by immunoblotting using the indicated antibodies. A representative result is displayed in (H). The p14ARF signals in the overexpression conditions were densitometrically quantified and normalized to the respective GAPDH signal, as specified by the numbers below the blot, with the untreated condition (- GEM) set to 1.
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(B) (Top) Illustration of the reporter constructs. The dashed line indicates the FISH probe. FISH to detect mRNA distribution at 24 hr after transfection of the H1C-SL-Rb (cH1C) and H1C-SLD-Rb (pH1C) constructs. C/N ratios were quantified Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.
|
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B, C, Changes in (B) length and (C) weight over time (Δ1 +/+ n=10, Δ1 -/- n=10, Δ2 +/+ n=10, Δ2 -/- n=12-13.
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(B) T47D-MTVLcells were untreated (0) or treated for 6 hours with 10 nM R5020, then the cells were lysed and total RNA was prepared, cDNA was generated and used as template for real time PCR with specific primers for BCAS1, KRT23 and IGFBP5 genes. The values were normalized with GAPDH and represent the mean and standard deviation from 3 experiments performed in duplicate. * P-value < 0.05.
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B) Estimation of the number of cells of each cluster per larva in normal condition (NI, orange) and after wasp infestation (WI, blue). For each cluster, the cell number was deduced from the total number of plasmatocytes and lamellocytes numerated and the proportion of each cluster in the singe cell datasets.
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J, K. Graphs represent the latency (sec) of the mice (WT and rescued mice at 8-10 weeks) to fall from the rota rod. WT (females, n=7, males n=6) and KO+AAV-Wwox (females n=7, males n=5).
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Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=54.22, LoD (RBD)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD.
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(C) Schematic representation of RHD domain location in p65 peptides (amino acids 19-306).
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(I) qRT-PCR analysis of Acta1 and Nppa in NRVMs. n=4:4. Data represent three independent experiments. Two-way ANOVA followed by Bonferoni post-tests was used for statistical analysis
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Diauxic lag times were fit from the monoculture data and are plotted as circles. Lag times could not be fit for conditions with too little growth on the remaining glutamate. The lag times used in the modeling are shown as lines through the data.
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A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.
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A. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on control plates and root lengths were measured 10 days after germination.B. The wildtype, ku70, the double mutants cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70 were grown on plates containing 0,6 μg/mL BLM. Root lengths were measured 10 days after germination.C. Graph represents the ratio of the mean growth rate on 0,6 µg/mL BLM compared to control experiments on plates lacking BLM for the wildtype, ku70, cycb1;1 ku70, cycb1;2 ku70 and cycb1;1cycb1;2 and the triple mutant cycb1;1cycb1;2ku70.
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A Left, Body weights of male WT and FADD-D mice on either a SD (n = 8 for each genotype) or a HFD (n = 6 for each genotype). Right, Food intake per mouse fed a HFD measured over 20 days normalized by body weight (n = 10 for each genotype). Results are means ± SEM. *P = 0.0007 (Student's t-test).
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Rose plots obtained with MDA-MB-231 derivatives exposed to an EGF gradient (shown as a blue and green wedge) in chemotaxis chambers. Blue lines indicate properly oriented tracks. Histograms (mean ± SEM) show quantification of persistence and chemotactic orientation from three experiments (45 cells).
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D. Netrin-3 silencing delays or inhibits SCLC engraftment in vivo. NMRI nude mice were subcutaneously engrafted with either NCI-H82-CRIPSR/cas9-NTN3 with three different guides RNAs (sG1 p=0.0022; sG2 p≤0.001; and sG3 p≤0.001) or control cells (sGc) n=6/group. Positive tumor engraftment was report when tumors reached 50 mm3 (Mantel Cox).
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a-c, hippocampal neurons were infected with rAAVs expressing GFP (control) or Npas4 (Npas4 overexpression) on DIV4. On DIV11, RNA was harvested and subjected to microarray transcript analysis. a, overexpression of Npas4 leads to ≥ 1.5-fold upregulation of the levels of 1091 sequences and to a ≥ 1.5-fold downregulation of the levels of 1133 sequences, indicating that Npas4 regulates a widespread transcriptional response. b, gene ontology analysis (GO) for biological processes via the Panther tool for up- and downregulated genes from panel a). Shown is a selection of relevant processes. Upregulated genes are enriched for cell-signaling processes (i.e., TGF-beta signaling, hormone responses), behaviour (i.e., locomotion) and developmental processes (i.e., anatomical structure development). Downregulated genes are enriched for synapse regulation (i.e., regulation of synapse assembly), dendritic organization (i.e. dendrite morphogenesis) and cognitive processes (i.e., learning, cognition, locomotion). c, selection of genes from the Npas4 regulon with relevance to synaptic transmission and dendrite morphology, both of which are processes implicated in habitual learning.
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