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B However, the absence of Atos leads to reduced levels of GR/HPR, LKR/SDH and Porthos. This decreases the OxPhos-generated ATP supply leading to defective tissue infiltration of the pioneering macrophages.
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K A549 cells treated with the indicated siRNAs were infected with S. pneumoniae R6 WT or ΔcbpF for the indicated durations. The cells were fixed and stained with DAPI and an anti-Atg14 antibody, and percentages of perinuclear-localizing Atg14 containing cells were quantified.
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(C) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice. Representative images of proximity ligation signals in 11 week old FUS and non-transgenic (NTg) littermate mice. Data were analysed by unpaired t-test; N=40 cells from 3 FUS and 3 non-transgenic littermates (age 10 weeks).
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(H) Immunoblotting of pS6 (Ser235/236 and Ser240/244), total S6, pAkt (Ser473) and total Akt in cell lysates from primary human keratinocytes treated with different doses of LL37 (0 - 8 μM) and scramble LL37 (8 μM) for 2 h. Scr, scramble LL37. pS6 and pAkt protein levels were analyzed relative to total S6 and Akt respectively. Tubulin is the loading control. Data (H) are representative of at least 3 independent experiments.
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F. Microscopy of the control, NDIME-/-, NDIME-/-+Rs26-Exon123, and NDIME-/-+Rs26-Exon4 cells at day 5 of neural differentiation. Scale bars, 100 µm.
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Histograms of RNA ISH analyses by RNAscope to quantify the level of expression of ITGA5 (D), DCN (E), PKD1 (F) and PLXND1 (G), assessed by the number of mRNA spots present in LYVE1+ cells in the vicinity of HF in P55 and P70 mouse back skin. n= average of 30 LYVE1+ cells / sample, n= 3 mice.
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G Twi1p was immunoprecipitated with an anti‐Twi1p antibody from wild‐type (WT) and COI12 KO (KO) cells at 2 hpm. Co‐precipitated RNAs were separated on a denaturing gel and visualized by the nucleic acid dye GelRed. The position of scnRNAs is marked with an arrowhead. The sizes of RNA markers (M) are indicated at the left. Precipitated Twi1p was analyzed by Western blot using an anti‐Twi1p antibody (bottom).
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(A) Laser injured DRG axons showing growth cone regeneration (upper panels) and regenerative failure (lower panels). Arrows as indicated.
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C. WT, 53bp1−/−, and Shld2−/− mice were immunized with NP-CGG and the serum was withdrawn 2 weeks post immunization and serial dilutions were subjected to ELISA analysis for NP-specific antibodies of the indicated isotypes. Values are mean absorbance ± SD of 4 biological replicates.
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Agonist-induced phosphorylation of ERK1/2 in HEK-293 cells expressing either V2R or B2R in the absence (control; CTL) and presence of βarr1 knock-down (βarr1-KD) are measured using Western blotting. Densitometry-based quantification of data (mean ±SEM) from four independent experiments is presented as bar-graphs in the right panels, normalized with respect to maximal signal under control condition (treated as 100%), and analyzed using Two-way-ANOVA with Bonferroni multiple comparisons test (***p<0.001, **p <0.01).
|
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E, Knockdown of endogenous PTPN3 promotes the Smad7-TβRI association. HEK293T cells were transfected with the indicated plasmids and treated with MG132 (20 µM) for 4 h before harvested. SFB-Smad7 proteins were retrieved with the Streptavidin pulldown. Levels of TβRI and other proteins were examined by Western blotting with the indicated antibodies.
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Relative mRNA levels of intestinal stem cell and differentiation marker genes in organoids described in (D). Data are represented as mean ± SEM. 4 biological replicates. *p<0. 05, **p<0. 01, ***p<0. 001 (t-test).
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two datasets (GSE110147, GSE32537) from lungs of patients with idiopathic pulmonary fibrosis (IPF) compared to normal controls (Ctrl). AU, arbitrary units. The number of patients per group is indicated.
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A HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm. B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.
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qPCR analysis of Ifnβ (A), Ifnα1 (B), Ifnα4 (C), Ifit1 (D), Rig-I (E), Cxcl10 (F) mRNA in WT or Sirt5-deficient BMDC cells (Sirt5 +/+ or Sirt5 -/-) infected with or without SeV (Sev or UI) for 8 h. Data information: UI, un-infected. The graphs represent fold-induction relative to the untreated cells. All data are presented as the mean values based on three independent experiments, and error bars indicate S.E.M. (unpaired two-tailed Student's t test).
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Proliferation of retinal ECs. H. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs, at P6.
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Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).
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(G) Decreased formation of mCherry-Atg8a-positive autophagosomes in SH3PX1 mutant clones (GFP negative, outlined) in fat bodies from larvae starved in 20% sucrose for 3 h. Bars, 20 µm. (H) The graph shows quantification of total mCherry-Atg8a spot intensity per cell for WT and SH3PX1 mutant cells. The graph shows mean ± SEM (error bars); ***, P < 0.001. Genotypes: (F and G) hs-flp; Cg-GAL4 UAS-mChAtg8a/+; FRT80B
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siRNA knockdown efficiency of Arl13b (E) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH
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GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.
|
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(E) Quantification of CD31+ area in heart sections of mice kept on a ketogenic diet or control diet for four months. Data are presented as mean ± SD. n≥3; *, p<0.05; **, p<0.01; ***, p<0.001 unpaired Student's t-test.
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Principal components analysis of transcriptional profile similarity for individual and hybrid lysates with different mixing ratios for (c) E. coli + C. glutamicum hybrids.
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(d) Venn diagram of the number of proteins identified from the different yeast strains.
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C ESI‐MS analysis of pure Ub (left, mass 8,565.65 Da, reference 8,565.80 Da) and purified phosphoUb (right, mass 8,645.61 Da, reference 8,645.80 Da). The difference of 80 Da corresponds to one phosphate group.
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B. EP300 acetylates H3K14 in PC3 cells. Western blot showing H3K14ac levels in PC3 cells treated for three days without and with the selective catalytic EP300 inhibitor A-485 (10 μM). Histone H3 serves as protein loading control.
|
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D IHC of SCA8 human cerebellum shows that PolySer accumulates in the white matter but not in the Purkinje cells (left panels) while polyGln accumulates in Purkinje cells but not cerebellar white matter (right panels).
|
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(F) HOIP-, HOIL-1L- and SHARPIN-specific mRNA is decreased in R6/2 striatum. Quantification of mRNA from the striatum of 9-12 weeks old R6/2 mice and non-transgenic littermates. Data are displayed as mean ± SD with n = 6-9 biological replicates from unpaired two-tailed Student's t-tests. *p ≤ 0.05, ***p ≤ 0.001.
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B: OSCC grade wise upregulation of ELDR expression was noted in the patient samples (N=20). Correlation analysis showed signification positive correlation of ELDR expression with tumor grade (R2= 0.926, significant P value = 0.01). Pearson correlation coefficient and p value was calculated using socscistatistics.com. Well: well differentiated, Mod: moderately differentiated, Poor: poorly differentiated carcinoma. Data are represented as the mean ± SD,
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(D) Reduction of debcl, Buffy, or p53 expression by RNAi resulted in a decrease in LTG fluorescence levels. (debcl, P = 0.018; Buffy, P = 0.006; and p53, P = 0.004).
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J. mRNA levels of indicated genes in isolated crypts from wild-type (n=4) and Angptl2-/-(n=4) mice following DSS treatment for 6 days and then untreated water for 6 more days (at days 12).
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Read distributions of RNAPII-CPSF160-tRIPs generated from Fus-silenced cells (siFus, pink line) and those of control siRNA-treated cells (siCont, green line). The p-values for the differences between siFus and siCont are indicated by circles. An arrowhead indicates a peak before APA sites in RNAPII-CPSF160-tRIP of siFus-treated cells.
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Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal.
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(F) Confocal microscopy of HeLa cells expressing GFP-Sec22b were treated with LLOMe, and stained for FTH1. Line tracings correspond to arrows. Scale bars, 5 μm;
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(D) MLEC from wild-type (upper panel) and EVL-/- (lower panel) mice stimulated with 10 ng/ml VEGF were stained for actin (cyan) and EVL (magenta). Asterisks indicate focal adhesions, white arrows indicate filopodia and white arrowheads indicate the leading edge of lamellipodia. Representative images from four independent experiments are shown. Scale bar, 20 µm.
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Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.
|
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(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).
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D. Decay rates of 256 different substrate RNAs. Data shown in (C) was normalized to overall decrease in substrate abundance as determined by phosphorimaging (as shown in B) and fit to the indicated model for exponential decay. Apparent decay rate of all substrates, as determined by phosphorimaging is indicated in red.E. Selected examples for individual substrates shown in (D). Values report the observed decay rate (kobs) in min-1.F. Overview of decay rates (kobs) of all 256 different substrate RNAs, as determined in (D). Error represents SEM of curve fit.
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D. Pearson correlation heat map of BAZ2A-bound regions and histone marks. Data for BAZ2A, H3K4me3, H3K27me3, H3K27ac, H3K14ac are from this work. H3K9me3, H3K9me2, H3K4me1 were obtained from ENCODE.
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FLT3 signaling was detected by Western blotting in zebrafish embryos-injected with FLT3/ITD mRNA (J).
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(H) Quantification of the viability of sensory neuron after 48h, 72h and 96h of infection. Sensory neuron viability was calculated as the percentage of transfected sensory neuron (GFP+/Tom+) in total sensory neurons (Tom+). For each time point, four biological replicates were included for statistical analysis. *P<0.05, **P<0.01, ***P<0.001, two-way ANOVA, error bars indicate SEM. "ns" is the abbreviation for "not significant". For all of the groups, at least 10 fields of the slides were included.
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(A) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture.
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G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.
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I-K. Adeno-associated virus overexpression of NDIME in the dentate gyrus (DG) of the dorsal hippocampus of VPA-treated mice rescued autism-like social deficits. Open field test (I), three-chamber test (J), and elevated-plus maze (EPM) test (K). Data in (I-K) are represented as mean ± SEM; n = 9 per group. Asterisks indicate a difference between VPA+AAV-GFP and Saline+AAV-GFP, whereas hash tags indicate a difference between VPA+AAV-NDIME-GFP and VPA+AAV-GFP. #P < 0.05, **/##P < 0.01, ***P < 0.001 (one-way ANOVA with Bonferroni post-hoc test). NS-non‐significant.
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The representative FACS plots show the frequency of CD31+CD34+ EPCs in the GFP+ population at day 5 with 1.25 nM PLB supplementation alone or in combination with NUMB_S ectopic expression from day 2.5. The bar graph denotes the percentage of CD31+CD34+ in GFP+ cells as described in (F). P-values were determined by an unpaired two-tailed Student's t-test.
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Hinge 1 moves away from hinge 2 during transition from pre-SC (cotton) to post-SC (lime), thereby forming new interactions with the ribosome. In contrast, hinge 2 movement from pre- (moss) to post-SC (emerald) does not change the interaction with the ribosome. The HLH motif is displaced from h5 in the pre- (watermelon) to h15 in the post-SC (pink). Positioning of the FeSD (sage) interferes with the closure of NBD2 (blush) in the pre-SC (rose). Possible communication pathways from ribosome binding sites to the NBSs in the post‑SC. HLH is connected to the Q-loop of NBSI via β8. I304 of hinge 1 connects to α14 which is adjacent to the His-switch in NBSI. Analogously, hinge 2 binding to the SSU might be communicated via Y593 and R566 to α23 next to the His-switch of NBSII. Interaction pattern of the communication pathways between HLH and hinge 1 to NBSI as well as hinge 2 to NBSII is different in the pre-SC compared to the post-SC.
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B | hFwe-Lose expression is elevated in nasopharyngeal swab probes from patients with comorbidities. Box plots illustrate an increased relative expression of hFwe-Lose in nasopharyngeal swabs of patients with diabetes (n = 129), COPD (n = 20), obesity (BMI > 30; n = 152), cardiomyopathy ("CM", n = 19), heart failure ("HF", n = 35), hypertension ("HT", n = 121), chronic kidney disease ("CKD", n = 60) versus disease-free patients (n = 96). Two-sided Student's t-tests were performed (compared to disease-free patients), and p-values are presented on the plot. The vertical axis represents relative hFwe-Lose expression normalized to the mean of non-hospitalized patients. The color refers to the COVID-19 disease outcome: gray for not hospitalised, blue for hospitalised and red for deceased patients. The shape of data points reflects the cohorts: circles for the training cohort (n = 203) and triangles for the validation cohort (n = 80). The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.
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(E) HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red) and DNA (blue).
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(E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1
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(A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B).
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HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).
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B, C Interactions of eIF3a, eIF3c and eIF3d with the ribosome: (B) shows an overview of the structure and zoomed views highlighting the interactions of eIF3a, eIF3c, the eIF3d N-terminal tail and the eIF3d cap-binding domain with the 40S, (C) shows molecular details of eIF3a interacting with eS1; eIF3c interacting with rRNA h22 and eIF3c and the N-terminal tail of eIF3d with the Zn-knuckle domain of eS27.
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EMSA showing that MYB73/MYB77 binds to the promoter of IAA19. Cold IAA19p or IAA19mp (MBSI motif mutated) was added as a competitor.
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A Bar plot showing the number of DEGs (adj. p-value <0.01, FC +1.5 in red, 1.5 < FC >-1.5 in white, FC< -1.5 in blue). Sorted B220+GFP+ spleen B cells from MaxKO-cd19, MycKO-cd19, DKO-cd19 and control HET-cd19 mice were activated with LPS plus IL-4 for 72 h and analyzed. Two samples for each condition were used.
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STAT1 phosphorylation and (G) STAT2 nuclear translocation in BTV-NS3-transfected Vero cells detected by immunofluorescence and confocal microscopy after 30 min IFN treatment. (Scale bar = 20 µm). Arrowheads in merge images indicate examples of transfected cells.
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U2OS wild-type cells were treated with a custom made anti-cancer library previously described Bezu et al, 2018() at 3 μM, supplemented with 500 μM oxaliplatin (OXA), 150 μM cisplatin, 50 μM resveratrol and 50 μM spermidine. For assessing transcription, cells were pre-treated for 1.5 h with the library followed by 1 h with the same drugs in which EU was added. The correlations between transcription inhibition and CALR exposure Known immunogenic drugs are indicated with colors: dactinomycin (DACT), mitoxantrone (MTX), doxorubicin (DOXO), daunorubicin (DAUN), OXA, docetaxel (DOC), paclitaxel (PACL), vinblastine (VB), vincristine (VC) and vinorelbine (VR)
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Male C57BL/6 wildtype (WT) and iNOS knockout (KO) mice were intraperitoneally injected with 1mg kg-1 LPS or an equivalent volume of carrier solution. Control WT, Control KO, and LPS-treated KO cohorts were pair-fed (PF) to the WT LPS-treated cohorts. After 18h, mice were euthanized, and tibialis anterior muscles were imaged by transmission electron microscopy. (left) Representative micrograph of tibialis anterior muscle (n=2). Scale bar = 1 µm. (right) Zoomed section of representative image to highlight mitochondria. Scale bar = 0.5 µm.
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(d; n = 3 independent experiments from 3 separate cell cultures and transfections; P = 0.933; F(5,12) = 0.249). HA-GluN2Bct-CTM-mediated cDAPK1 knockdown was significantly reduced by NH4Cl (c; 20 mM; n = 4; ΔΔΔP 0.001 compared to HA-GluN2Bct-CTM:cDAPK1 8:1 group) and by mutational inactivation of CTM
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A, B Representative transmission electron microscopy (TEM) images showing keratinocyte mitochondria (red arrow) in patient 1 (A) and in the Mbtps1-conditional knockout (cKO) mouse model (B). Scale bars: 2 µm.
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Quantification was performed to determine number of positive vesicles per cell (ImageJ software). Nuclei are shown in blue (DAPI). Data represent mean ± SEM of cell fields randomly selected (n=13-20 for (C) and n=11-20 for (D)). Data are collected from at least 3-4 independent experiments. For violin plots (D), thick lines represent median and thin lines interquartile; ***p<0.001; **p<0.01; *p<0.05, unpaired t-test or Mann-Whitney, as accordance.
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A. H3122 cells expressing endogenous EML4-ALK V1 and HEK293 transfected with YFP-EML4-ALK V1 WT and KD were stained for either anti-ALK or anti-GFP (green), anti-α-tubulin (red), and DAPI (blue). Scale bars, 10 μm; magnified views of a selected area are shown. B. Violin plots showing the number of EML4-ALK V1 cytoplasmic foci per cell. Data represents 30-50 counts from three biological replicates. ***P<0.001, ****P<0.0001 in comparison to HEK293 YFP-EML4-ALK-V1 KD by one-way ANOVA analysis.
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H-K. TEM images of 5 week old adult retinas corresponding to (H) wildtype; (I) ADAM17-/- mutant; (J) glial overexpression of lipase in wild-type glial cells; (K) neuronal overexpression of lipase in the ADAM17-/-mutant. Scale bars: 10μm.
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C. KT250 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-WT) and KT235 (ΔS/O/H/M ssk2/22Δ hog1Δ STE11-Q301P) were transformed with pRS416-Hog1 (WT) or pRS416-Hog1-N149H D162G (N149H D162G) together with pRS414-8xCRE-lacZ. Expression of the Hog1 reporter gene 8xCRE-lacZ in the absence of osmostress was assayed. Data information: (C) Error bars are SEM (n=4).
|
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A: Serial dilution assay of yeast strains with different Duo1-6xFlag alleles in a wild type or Dad1-GFP strain background. Growth phenotypes of the respective yeast strains were analyzed by spotting serially diluted amounts of cells on YEPD plates and incubation at different temperatures.
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(C) to (E) The contents of free IAA (C), IA-Asp (D), and IA-Glu (E) in the root tips of wild type and the kup9-1 mutant. Data are means ± SE (n = 3, biological replicates; each replicate contains 1000-1200 individual plants). Student's t-test (*P < 0.05, **P < 0.01) was used to analyze statistical significance, and "#" represents control.
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Evaluation of TGFβ signaling activity in mouse colon tumors after four consecutive daily injections of hosts with 40mg/kg of the MMP2/9-inhibitor SB3-CT (MMPi). Quantification of pSMAD3+ cells (left panel) and pSMAD3 staining intensity per pSMAD3+ cell (right panel) on at least five arbitrarily selected tumor areas per section of MMPi (n=6) or vehicle (n=6) treated mice.
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E: Schematic illustration of the AdPROM-mediated degradation of FAM83D. VHL; Von Hippel Lindau protein, CUL2; cullin 2, RBX1; RING-box protein 1, E2; E2 ubiquitin-conjugating enzyme, aGFP.16; anti-GFP.16 nanobody.
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Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).
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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. C. Bar plots showing significantly enriched gene ontology (GO) terms scaled by Log10(P-value). Red GO terms are upregulated and blue GO terms are downregulated in symptomatic P. falciparum malaria compared to healthy immune controls.
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(D) T-LipoAS plasma disappearance rate in murine ear veins and estimated half-life obtained by intravital microscopy. The mean fluorescence intensity averaged between the remaining 24 regions of interest (from n = 4 FVB/N mice) and the positive standard error depicted in the figure.
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(b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane.
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E) RB levels of the cells after 14 h of the release of nocodazole treatment. Etoposide was used to induce genotoxic stress.
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C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition).
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(C) In vivo limiting dilution assays. miR-10-OE or control cells were subcutaneously injected in the backs of mice at 3 doses (1,000, 10,000 and 50,000 cells for MCF-7 and 5000, 10 000 and 20 000 for SKBR-3). Mice were sacrificed after eight weeks, and tumor burden was analyzed using the ELDA software. Plots are depicted in Fig EV2. The tables show the number of mice with tumors versus the total number of mice injected. Below the tables, the estimated frequency (freq) of stem cells for each group and p is shown. Stem cell frequency for MCF-7 cells was 1 in 34252 (range 94888-12364) in miR-10b-OE cells versus 1 in 238965 (Range 1817801-31414) in miR-scr cells. Stem cell frequency for SKBR3 cells was 1 in 5013 (Range 11135-2257) in miR-10b-OE cells versus 1 in 18778 (Range 45397-7767) in miR-scr cells.
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E. Heatmap of transcript abundances for individual genes (rows) representing 12 expression modules detected by EBseqHMM filtered for genes with a significant GXS glm interaction. Genes with observed functional similarity within modules are highlighted. Nr5a2, Lifr, and other pluripotency genes shared the same expression path (m4) that decreases more significantly in B6, retained expression in D2, when ESCs are differentiated to EpiLCs. Sox1, Pax6 and other neurogenesis genes shared an expression path (m5) that is significantly up regulated in B6 in EpiLCs. Gstp2, Smarcd3 and other cell cycle regulation genes shared an expression path (m9) that is significantly up regulated in B6 ESCs.
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(F) RNA immunoprecitation (IP) with anti-GFP antibody in wild-type (mock IP) and nos-Gal4/UASp-GFP-Aub ovarian extracts. Cbl mRNA was quantified using RT-qPCR. Normalization was with RpL32 mRNA. Mean of three biological replicates. The error bar represents standard error to the mean. * p-value <0.05 using the two-tailed Student's t test.
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myogenin and MyHC in C2C12 myocytes transfected with siRNA targeting mGPDH. Quantificatio represents the levels of indicated protein normalized to β-actin at indicated day after differentiation
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F. Number of visits to the platform during the probe test. young-control (5.2 ± 2.5, mean ± std. deviation); old-control (2.7 ± 1.83, mean ± std. deviation); old-inhibitor (5.0 ± 2.3, mean ± std. deviation). All mice were male. Number of mice: young-control, n = 18; old-control, n = 18; old miR-inhibitor mix, n = 20.
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(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.
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(B) Recruitment of endogenous SLFN11 to laser-induced DNA damage sites. 40 min after laser irradiation, cells were stained with antibodies against SLFN11 and RPA2. Scale bar, 10 μm.
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A. HEK293 cells were treated for 8 and 24 h with 100 μM H2O2. GSNOR was evaluated by Western blot. Densitometry of each lane is normalized to GADPH, selected as loading control, and expressed as arbitrary units. Values shown represent the means ± SD of n ≥ 3 independent experiments. *, p<0.05; **, p<0.01 calculated with regard to H2O2-untreated cells.
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(D) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml), zVAD (20 μM), Nec-1 (40 μM), and GSK872 (10 μM), as indicated, for 8 h. Cell viability was determined by ATP assay. Data show mean ± SD of three biological replicates. (E) WT and Pml-/- immortalized MEFs were treated with TNF (100 ng/ml),IKK inhibitor BMS-345541 (10 mM) and Nec-1, as indicated, for 6 h and then cell death was assayed. Data show mean ± SD of five biological replicates, except the Nec-1-containing experiment (n=2).
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Histograms derived from the curves presented in (A) and presenting apparent dissociation constants (Kd) for each Siz2∆CT variant.
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A. Overexpression of cortactin compensated for the knockdown of KV10.1 in ciliary disassembly (4 h serum reintroduction after starvation).
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(D) left: cartoon representation of the NHE9 ion-binding site in the 6-TM core transport domain, which is made up of two broken helices TM5a-b (green) and TM12a-b (purple) accessible to a sodium ion from the cytoplasm (yellow sphere). right: ion-binding site residues are shown as yellow sticks and labelled with the corresponding residues in PaNhaP (PDB id: 4cz8) shown as grey sticks.
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G-H: Cells with the indicated genotypes carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for one hour before filming. Cells were filmed at 34°C every 2 or 4 minutes by time lapse fluorescence microscopy. Chromosome V segregation errors are reported in the table (H). Representative cells are shown as examples in the montages (G). Representative montages for wild type and mps1-3 cells are shown in Fig. 1F. DIC: differential interference contrast. Scale bar: 5 μm.
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16-days old MCF10A spheroids grown in matrigel/collagen mix were stimulated for 24h with either macrophage-conditioned or control medium. Macrophage-conditioned medium induces invasive protrusions in MCF10A spheroids compared to control. Error bars represent mean ± SEM from 11 independent experiments (n=2 per condition; at least 15 spheroids each from at least 2 fields of view). Macrophage donors are indicated as D1-D16. M1D - M1 differentiated, M1A - M1 activated, M2D - M2 differentiated, M2A - M2 activated macrophages.
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(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.
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(d) Effect of gp91 ds and PP2 on LC3-LAMP1 colocalization was analysed using confocal microscopy. Representative images from independent biological experiments (n=3) are shown (scale bar=140 and 40 μm for red box areas).
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Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.
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G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC-MitoT, PBMC and non-transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells.
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B. Northern blot analysis of substrate tRNAs of hTrmt13 from control and the hTrmt13 knockdown MDA-MB-231 cells.
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B. Flag-tagged INVS interacted with HA-tagged Akt1 (lane 1-3), Akt2 (lane 4-6), and Akt3 (lane 7-9) in co-immunoprecipitationassays. Expression of each Akt isoform and INVS were similar.
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Oxygen consumption in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).
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Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.
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Reactome Pathway Analysis of the differentially expressed genes at 10% FDR after 12 weeks of treatment (see Expanded View document for details).
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I Schematic showing ketone metabolism pathway (left panel). Reversible enzymes involved in the production of acetyl-CoA, acetoacetyl-CoA, acetoacetate, βOHB, mT, SCOT1/2, and BDH1/2 are indicated in bold case. Immunoblots of ketone metabolic enzymes (BDH1/2, SCOT1/2, mT) (right panel) in pancreatic tissues from 7-week old KI (n=3 for BDH1/2, SCOT1/2, and n=4 for mT) and KIC mice (n=4).
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C, DAverage relative intensities over time and corresponding fluorescence t 1/2 values of heterochromatic Swi6 obtained from FRAP experiments performed with wild‐type (brown) or cid14∆ (dark green) cells expressing Swi6‐EGFP.
|
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] |
(F) p16-3MR mice were treated with abemaciclib (50 mg/kg, 7 consecutive days) ± pifithrin-α (2 mg/kg, 7 consecutive days). Values indicate the ratio of post-treatment bioluminescence to pre-treatment (n=5 mice/group). Data information: Data are means ±SD. Unpaired student t-test. *p<0.05, **p<0.01, ***p<0.001, N.S.=not significant.
|
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(C-D) C57BL/6J mice received control or 25 mM ZnSO4 drinking water for 7 days. Then semi-solid ileum slurry was isolated, weighed, mixed, diluted and normalized and was plated on TSA plates. After randomly picking 110 and 102 colonies from plates of H20 treated mice and ZnSO4 treated mice resp., their identity was determined by MALDI-TOF and expressed in a pie diagram. Chi2-tests were performed.
|
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] |
C Immunoblot for IP3R type 1 (top) and IP3R type 3 (bottom) in DCs transduced with lentivirus encoding for different IP3R shRNAs. Actin was used as a control.
|
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