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(F) Protein synthesis assays using the Cayman kit and Coomassie brilliant blue (CBB) staining were performed in the indicated cells. WB was performed using the indicated antibodies (left), and the relative intensity of protein synthesis was quantified (right). Unpaired t-test was used. Data information: The data are represented as the means ± SEM of three independent experiments (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).
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E: Median shift in the CF of the area change rate and F (blue symbols, N=52 biological replicates), the LimE-GFP intensity (brown symbols, N=17 biological replicates) and the GFP-myo intensity (yellow symbols, N=7 biological replicates). Data below the dashed line indicates cells for which no significant correlation was found. Shift values are -11.2 (-15.0/-9.4) s, 11.3 (7.5/11.3) s, and -26.3 (-30.0/-14.1) s.
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(G) Immunostaining of cathelicidin in HaCaT keratinocytes treated with LL37 (4 μM) ± RAPA for 24 h. DAPI staining (blue) indicates nuclear localization. Scale bar: 50 μm. All results are representative of at least 3 independent experiments. Data represent the mean ± SEM.
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(B) Venn diagram showing overlap of differentially expressed (DE) genes between treatments (P-values < 0.05), bubbles are drawn to scale. A total of 1451 DE genes was found in comparisons between all treatments and relative percentages to this number are indicated.
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Expression levels of EMX2 (top) and EZH2 (bottom) in tumour or adjacent normal regions laser microdissected from human GBM tumours as detected by RNA-seq. Data sourced from the Ivy Glioblastoma Atlas. FPKM: fragments per kilobase of transcript per million. The significance of the differential expression in normal and tumour regions across patients is indicated (two-way ANOVA). Bars represent mean ± SEM. N: 3 regions sampled for each GBM tumour, 2, 3, 2, 3, 2, 3, 3 and 1 regions for normal tissue of patients 1-8, respectively.
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G Western blot analysis to detect cleavage of wild-type (WT) or modified (P259A) FdnH with active (+) or inactive (SAHA) Rhom7 expressed from pBAD33 with (+) or without (-) FdnI. rhomboid substrates that are uncleaved, cleaved by Rhom7, are marked by black, blue arrows, respectively.
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H. Kaplan-Meier curve of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or greatest dimension ≥ 1.5cm. Red arrow: start of therapy, day 1. ****p < 0.0001 (log-rank Mantel-Cox test)
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% of adult females that hatch after crossing female dTor+/- ;; dLap1-/TM6C with male y/FM7i ;; dLap1-/TM6C flies. Two-tailed T-Test. **** p < 0.0001, compared with expected Mendelian ratio (n≥3).
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D, Schematic representation of the AAV-dCAS system: For induction of up to five endogenous genes plus a GFP reporter, a total of four different AAVs are utilized. dCas9 is delivered by two AAVs, a third AAV is needed for the delivery of the SAM activator. A fourth virus contains the reporter gene, while sgRNAs are distributed between the vectors. To ensure cell type specificity upon Cre expression, the N-dCas9 and the GFP are inverted and flanked by two different LoxP sites (LoxX and Lox511). Data information: Abbreviations: dN-Cas9 - N-terminal dCas9-residues 1-573, N-intein - N-terminal part of DNA polymerase III subunit alpha, dC-Cas9 - C-terminal dCas9 residues 574-1368, C-intein - C-terminal part of DNA polymerase III subunit alpha, VP64 - 4x VP16 herpes simplex virus protein vmw65, p65 - p65 subunit of human NF-ĸB, HSF1 - heat shock factor 1, MS2 - MS2 bacteriophage coat protein, PAM - protospacer adjacent motif, TSS - transcriptional start site, CBh - chicken β-actin hybrid promoter. SgRNA expression is driven by the different Pol III promoters (H1, hU6, mU6 and 7SK).
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(A) Representative IHC images of CXCL12 intensity and staining pattern in the TMA. N = 219 tumors. Upper panel, scale bar, 400 μm; lower panel, scale bar, 100 μm. Arrows point at punctate staining in the stroma.
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(F) Under nutrient repletion conditions, almost no autophagic elements are detected in Epg5+/− MEFs. RER, rough ER; M, mitochondrion; N, nucleus. The arrowhead indicates a vacuole with a late residual body-like appearance (aAV-III). (G) Under nutrient repletion conditions, Epg5−/− MEFs accumulate a large number of autophagic vacuoles. Red arrow, a likely autophagosome; red arrowheads, complex vacuoles of the aAV-I type; white arrowheads, aAV-II vacuoles
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qPCR quantification of SOX1 in wildtype, PAX6 KO and PAX6D KO cell at day 20 of retinal differentiation (n=3 biological replicates for each sample).
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Measurement of L-amino acid or essential amino acids (leucine, isoleucine, valine) contents in Scgb1a1 lineage-labeled tdTomato+ organoids derived from control, Lats1/2 dKO or Raptor;Lats1/2 tKO mice. Data are presented as mean ± SEM (n=3 technical replicates for each genotype). *p<0.05, **p<0.001 (Student's t-test).
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(A) Schematic representation of the genomic locus of the chromosome 1 of V. cholerae with the three parS sites, named parS1-3. The rRNA operon (blue rectangle) spans the genomic coordinates 53823 to 59123 (B) ChIP-seq performed on strain N16961 is displayed as the number of nucleotide reads in function of the genomic coordinates. Correspondence to the parS1-3 location represented in (A) is indicated by grey dotted lines. The number of reads in the Input sample (grey line) is normalized to the total number of reads in the IP sample. (C) We modeled the ChIP-Seq data as in Fig.1C-E by means of MC simulations with a Freely jointed chain of size N=2000 monomers of size a=16-bp. Data are normalized after background subtraction to the read value at parS1 (genomic coordinate 62438). The best fit was achieved with σ=25nm and an amplitude κ=0.15 leading to Nt~50 ParB on the chromosome. In the MC simulation, we accounted for the finite width of the distribution around parS sites by including the average fragment size of the DNA librar
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(A) LM2 cells transfected with siRNA targeting PTPRN2, PLCβ1, or control siRNA were stained with phalloidin (red) and DAPI (blue) and analyzed using fluorescence microscopy. Right, mean fluorescence intensity quantification of whole cell phalloidin signal. N = 40 cells/group. Scale bar, 10 μm.
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MT repair sites on GaMTs and non-GaMTs extracted from C. Red: GaMTs; Green: non-GaMTs; yellow: GTP-tubulin. MT repair sites on GaMTs and non-GaMTs separately presented in detail. White arrows indicates GTP-tubulin labeling sites. Scale bar: 5 μm. Box-whisker plot presents the ratio of MT repair sites on GaMTs and non-GaMTs separately. (1 representative of 3 independent experiments and n= 8 cells). ***p < 0.001, unpaired t-test.
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Simultaneous expression of Y701F-STAT1 rescued the hTau-induced suppression of NMDARs protein (K) expression measured by Western blotting in hippocampal CA3 of C57 mice (n=4). Data information: Data were presented as mean ± SD two-way analysis of variance (ANOVA) followed by Bonferroni' s post hoc test *, p<0.05, **, p<0.01, ***, p<0.001 vs eGFP or hTau; #, p<0.05, ##, p<0.01 vs hTau.
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(d) Mitophagy was analyzed in untransfected (UT) SH-SY5Y cells or in stable Flag-Parkin overexpressing SH-SY5Y cells transfected with either scrambled (scr) or Fbxo7 siRNA. Histogram indicates the percentage of cells with no remaining mitochondria following 24 h treatment with CCCP (10 μM) for each condition. Complex Vβ subunit (CxVβ) was used as a mitochondrial marker. Data are presented as mean ± s.e.m., **P 0.01. Scale bars, 10 μm.
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B Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β-Actin was used as the reference.
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Confocal images and 3D reconstruction showing the intracellular localization of endogenous βCaMKII (green) in CAD cells. Cells were stained with DAPI (blue) and phalloidin (red). Yellow arrows point to βCaMKII-positive puncta inside a TNT. Scale bars in the expanded fields represent 10 µm in the micrographs and 1 µm in the 3D projection.
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Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Cell surface expression of hCD2 and intracellular staining of Bcl6 are shown for Prdm1ihCd2/+ (red) and wild-type (gray) GC B cell The absence of Bcl6 expression in non-GC B cells (black) is shown as reference
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Diploid (2n) and tetraploid (4n) strains expressing a Gam-GFP reporter were cultured on YPD or PRE-SPO medium at 30°C or 37°C, respectively, in the presence (Gam-GFP ON) or absence (Gam-GFP OFF) of doxycycline (DOX) for 24 h. Cell images indicate calcofluor white staining (cell wall; blue), GFP, and a merged image of GFP/DAPI/DIC channels. Scale bar = 10 μm.
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Covariate single-cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24-h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK-IN-8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively.
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F. Venn diagram illustrating the number of TSSs containing binding sites for Jmjd2a and/or Jmjd2c within +/- 1kb. The p value was calculated using hypergeometric test.
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(E) IP of β2AR in the absence (Control) or presence of 10 μM Pep2 or Pep2 with S1928 being phosphorylated (phPep2), which did not displaced α11.2.(F) α11.2 and GluA1 IB signals were normalized to β2AR signals (***p<0.001, ANOVA).
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(B, E, and F) At the indicated time points post-infection, WCL were extracted from HCV-infected Huh7 cells. (B) Parkin ubiquitination in WCL extracted from HCV-infected cells. All ubiquitinated proteins were immunoprecipitated with anti-Ub antibody and analyzed by immunoblotting with anti-Parkin antibody. HCV infection was verified by immunoblotting with anti-HCV core antibody. β-actin was used as an internal loading control.
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I - Left: Representative flow plots showing ChAT-eGFP+ and Cx3cr1CreER-RFP+ double positive MΦ (yellow gate) in the IWAT of ChAT-eGFP;Cx3cr1CreER-RFP mice. Right: Percentage of ChAT-eGFP+ MΦ that are RFP- or RFP+ in the IWAT of ChAT-eGFP;Cx3cr1CreER-RFP mice (n = 5).
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Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.
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(a) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl-2. HeLa cells, untreated or treated with 10 μM Y27632 (top panel), were cultured in control or glucose-free (4 h) medium, lysed and cell extracts prepared. Endogenous Bcl-2 was immunoprecipitated with cross-linked Bcl-2 agarose and resulting eluted immune complexes were resolved by SDS-PAGE and blotted against indicated antibodies. EJ shCont. and shROCK1#1 cells (bottom panel) were grown in control or starvation media for 4 h and Bcl-2 IP was performed as above. Inputs were rerun to confirm ROCK1 knockdown.
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G, H Reporter cells were subjected to nutrient starvation for the indicated time. AMPKα Thr172 and HSF1 Ser121 phosphorylation was measured by immunoblotting.
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(A) FAK−/− cells stably re‐expressing FAK‐wt, FAK‐Y397F, FAK‐KD or FAK‐Y4F‐Y9F were immunoblotted using anti‐FAK, anti‐FAK Y397, anti‐FAK Y861, anti‐FAK Y925, anti‐Src, anti‐Ret and anti‐actin. FAK and Ret were immunoprecipitated from these cells and then samples were immunoblotted with anti‐Src, anti‐FAK or anti‐Ret.
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G, H. RNase H2 enzyme activity is reduced in mouse and patient cells. (G) Enzyme activity for Rnaseh2bA174T/A174T MEFs and passage-matched Rnaseh2b+/+ controls, against RNase H substrate (RNA:DNA heteroduplex) and RNase H2-specific substrate, double stranded DNA with a single embedded ribonucleotide (DRD:DNA). Mean activity for three independent cell lines, error bars, SEM. Enzymatic activity expressed relative to the average value of control MEFs. *** = p<0.001, two-tailed t-test (n=3 Rnaseh2bA174T/A174T and n=3 Rnaseh2b+/+ control MEF lines). (H) RNase H2 activity in LCLs from two independent healthy controls and an AGS patient homozygous for the RNASEH2B-A177T mutation. Enzyme activity normalised to average activity of control lines. Three independent experiments, error bars SEM. *** = p<0.001 versus either control, two-tailed t-test.
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F) Three example traces from a single cell exposed to 405nm followed by 525nm light. Individual traces were fit with exponential decay (405nm, blue) or association curves (525nm, green) for 6 trials across 2 cells
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E No increased LDH release was observed after 14-day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC-MN lines (unpaired t-tests, Becker S6: significant decrease in LDH, **P = 0.0064; M211R2: not significant, P>0.05). In Becker S6, mean LDH release was 22% lower after H89 treatment compared to vehicle. Treatments were performed in eight technical replicates. Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01.
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(C quantification of the percentage of mitotic cells showing aberrant SC35 mitotic bodies is reported (C). Number of cells counted: 923 (control), 694 (GA), 951 (17AAG) and 1019 (VER). n = 6 independent experiments, ± sem, p < 10-10, ± sem (One-way ANOVA).
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(E) CD43-negative primary splenocytes from 8-week-old wild-type (WT) and Pogz+/∆ mice were stimulated ex vivo with IL-4 (50 ng/mL) and LPS (25 μg/mL). Cells were harvested at the indicated time points and assessed for their surface expression of IgG1 by flow cytometry. Data are represented as a bar graph showing the mean ± SEM, each mouse being represented by a round dot (WT) or a square (Pogz+/∆) (n=6 mice per genotype). Significance was determined by two-way ANOVA followed by a Bonferroni's test. *P<0.05, **P<0.0001.
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(B) HeLa cells stably expressing YFP‐Atg5 were treated as in (A) and monitored by live microscopy. Quantification of the lifespan of Atg5‐labelled puncta structures is presented. Mean±s.d. of three independent experiments is presented. *P0.05, **P0.001.
|
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K. Schematic of intermediates and extension products generated by PsiI+XhoI double digest of pICLpt.
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Representative images of recombined endothelial cells in transversal sections of the aorta. The distribution and amount of recombined endothelial cells is similar in IgG and αDll4 conditions. CD31 is shown in white in the image, in black in the label. Multistack reconstruction of confocal images. Scale bar: 50μm. Quantification of recombined cells in the aortic endothelium using confocal images. Mean±SE (n=3).
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E) qRT-PCR expression of endogenous repeats following KAP1 knockout in 293T cells Results were normalized to B2M. KAP1 expression levels were verified by qRT-PCR and Western blot. For (E), we found that clone I (also treated with KAP1 sgRNA) retained KAP1 protein expression (E, right panel) so we only used clones II, III and IV to explore phenotype (E, left panel), which represented validated KAP1 knockouts. Two-tailed unpaired t tests were done and p-values are: (E) HERVK14C_2: <0.0001.
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After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)
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Co-IP assay was performed to detect the efficiency of dimerization between SPOP WT and Q165P mutant (F)
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(E) The response of PI4K2A K/O cells was significantly reduced Means ± S.E.M. are shown from three experiments performed in triplicates.
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Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A).
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(D) Mean RLS in BY4741 wild-type, nat4∆, fob1∆ sir2∆ double and fob1∆ sir2∆ nat4∆ triple mutant strains.
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(C) ATPase activity of recombinant 6x-HisRCAß1 WT, K438Q and K438R with ATP and ADP/ATP= 0.11, respectively (n = 3, *t-test p-value < 0.05, +t-test p-value< 0.1).
|
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Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.
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(C) Effect of rISG20-Fc treatment on pregnancy loss. rISG20-Fc treatment significantly decreased the pregnancy loss caused by ZIKA infection. The pregnancy loss rate (number of dead fetus + resorptions/ total number of implantations x100) was calculated. Data represent as mean ± SEM; n=3-4 mice per group; *p < 0.05 and **p < 0.01 by One-way ANOVA.
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F. Electron microscopy pictures of chylomicrons obtained by ultracentrifugation of plasma from Pkd2wt/wt and Pkd2ki/ki male mice as specified in methods.
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(H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.
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Northern blot validation of mascRNA in the RIP samples. 5S rRNA from the RIP samples was used as an internal control.
|
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Expression of the indicated proteins in HEK293 cells was examined by western blotting, and β-Actin was used as loading control. This summary is a representation of three independent experiments.
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(F-I) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the tRNA levels (y-axis, Each dot represents a single codon. Graphs for all codons (F), codons for polar amino acids (G), nonpolar amino acids (H), and charged amino acids (I) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.
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(B) Immunoblotting of whole cell extracts obtained from parental and RAP80-/- clones in the RPE-1 hTERT p53-/- Cas9, RPE-1 hTERT p53-/- BRCA1-/- Cas9, U2OS, U2OS-2-6-3 and U2OS Flp-In/TREx cell lines with RAP80 antibodies. Tubulin is used as loading control. Representative of at least two independent immunoblots. See Figure EV1 for further details on the gene editing strategy used to knockout RAP80.
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C, Schematic representation of three classes of TSN-interacting proteins, i.e. stress-dependent, stress-sensitive and stress-independent interactors, identified upon the comparison of TSN2_NS and TSN2_HS protein pools.
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C. NMD can occur in UPF3A/3B-independent manner. It remains possible that the EJC can still communicate with the premature termination complex in a UPF3A/3B-, and possibly UPF2-independent manner (indicated by a red dotted line with a question mark) to elicit NMD. mRNAs are targeted to NMD at a lower probability in the absence of UPF3 paralogs (signified by a lower weight arrow).
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(C) Representative western blot of ORF65 levels in scr and circHIPK3 KD cell lines with GAPDH as a loading control. Densitometry analysis of n=3 western blots. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
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Kaplan Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log-rank test. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.
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(a) HeLa cells constantly expressing HA-tagged PML were depleted for endogenous SUMO1/2/3 by siRNA. Subsequently, they were transfected with RGS-His tagged SUMO2T90K for 72 h. Prior to lysis, cells were treated with As2O3 for 2 h. HA-tagged PML was immunoprecipitated via a denaturing HA-IP, which was followed by LysC digestion. Peptides harboring GlyGly-remnants, corresponding to SUMOylation sites, were enriched by GlyGly-IP and the samples were analyzed by LC-MS/MS.
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Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.
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(E) Transiently expressed Flag-N-HttQ68 and myc-LC3 colocalize in GFP-p62-positive structures. S-GFP-p62 cells were fixed 48 h after transfection, Flag-N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP-N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.
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Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.
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E-F. Schematic representation of the promoter fragments from the MCIDAS (E) and FOXJ1 (F) regulatory elements assayed by chromatinimmunoprecipitation. For FOXJ1, two putative transcription start sites (TSS) have been proposed and are indicated (see materials and methods).
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C The addition of 20 nM queuine to the SF medium is sufficient to restore tRNAHisQUG in HeLa, whereas 200 nM of q is necessary for queuosinylation of tRNAHisGUG in HCT116 cells. Data information: S (standard medium), SF (serum free medium), q (queuine), Q (queuosine), G (guanine).
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Immunoblot analysis of WT, caspase-1 KO (G) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.
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Predicted structure of UBR7 PHD (a.a.133-190) using Phyre2 (Kelley et al, 2015) overlayed on the BPTF PHD2 structure (RSCB PDB: 2F6N) as reference. UBR7 PHD aromatic cage residues mutated in (D) and corresponding residues in BPTF PHD2 are labeled.
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I Confocal fluorescent images of 4‐fold mtm‐3 embryos expressing ATG‐18::GFP (I′) and stained by DAPI (I) and anti‐LGG‐1 (I″) antibody. Insets show magnified views. Arrows indicate ATG‐18 puncta labeled by anti‐LGG‐1 antibody. Scale bars: 5 μm.
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A portion of transduced HSCs were plated into CFU assays, incubated for 12 days and CFU colonies analysed. intracellular human IL-1Ra were quantified (n=6 CFU assays).
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(G) EGFR was not required for ligand-induced phosphorylation of STING Ser366: EGFR-/- HeLa cells or gefitinib-treated and untreated WT HeLa cells were treated with cGAMP for the indicated time and cell lysates were analyzed by Western blot.
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A 35S-labelling of newly synthesized mitochondrial-encoded proteins of RKO cells treated either with DMSO, IMT1, rapamycin and FG4592 alone or in combination with IMT1. Left: pulse labelling; right: 24 hours chase in normal medium. Putative mitochondrial proteins are reported on the right-hand side of the gel, molecular weights on the left. Coomassie staining shows equal protein loading; the image is representative of n=4 independent experiments.
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(O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).
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Table of differentially expressed genes (DEGs), in LV during late Telogen compared to mid Telogen, relative to biological processes, analyzed using the Enrichr platform (Chen et al., 2013; Kuleshov et al., 2016). Upregulated (red), downregulated (blue). Data shown represent the fold change of the genes.
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qRT-PCR analysis of effect of ZNF596 knockout on EZH2 expression. Data information: data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by One-way ANOVA
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Cryo-EM visualizes tRNA-bound nascent chain (red, NC) within the ribosomal tunnel (A) and a density between separated TMHs 2b and 7 of the lateral gate (B, shown in mesh). The proximate cysteine positions within SecY used for cross-linking are indicated and highlighted in red.
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(K) F-actin levels assessed by FACS sorting of labelled MIN6 cells transfected with siRNAABCA12 (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test).
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E Co-immunoprecipitation of NB-3 and PTPσ from sensorimotor cortex lysates of NB-3+/+mice after SCI.F Quantification of immunoprecipitated PTPσ by NB-3 in (E). **p < 0.01; one sample t-test. Data were analyzed from 3 independent experiments each including 15 mice per group.
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Venn diagrams show the numbers of up- and down-regulated introns upon YTHDC1 KD (|logFC|≧1, p<0.01) in HSATIII-expressing cells (magenta) and/or HSATIII-depleted cells (cyan).
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(A) Analysis of SODG85R‐GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70‐transfected HEK cells. Representative merged images of cells with cytoplasmic, pre‐aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3‐positive autophagosomes. Scale bar, 5 μm.
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Comparison of the effect of MMAF and EV20/MMAF on the proliferation of BT474 and BTRH cells after five days of treatment with the drugs.
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A, HeLa cells treated or not, with 2µM Mito-C for 15 min were immunostained with anti-TOM20 (red) and anti-DRP1 (green); arrowheads in the far-right panel indicate recruitment of DRP1 onto the mitochondrial surface (TOM20). B, Quantification of DRP1 signal on TOM20 positive structures. Errors bars show the standard error of the mean (SEM) (n=45, technical replicates). C
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(B) The characteristic ubiquitylation ladder was observed for wild-type MITOL, but was absent in the K268A and K54A/K268A mutants. Black arrowhead indicates 3Flag-MITOL.
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(A) Cryo-EM reconstruction of the eEF2 bound 80S ribosome adopting a rotated POST-1 state bearing chimeric ap/P- and pe/E-tRNA as well as a disordered eEF3.
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qPCR analysis showing the relative expression of ECM marker genes in obese (HFD 16 weeks) eWAT of WT (blue bars) and CD169-DTR (red bars) mice (DT treatment for 7 days). n = 4-6 mice per group, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
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a, RAW cells expressing GFP-LC3 were transfected by electroporation with control or Atg5 siRNA oligonucleotides. At 24 h after transfection, cells were fed with zymosan (yellow arrows) and the internalization process was followed by time-lapse video microscopy. Images obtained at 5 and 30 min after zymosan engulfment are shown. b, The percentage of GFP-LC3-associated phagosomes (n ≥ 100 per /group) was obtained from three independent time-lapse videos (2 h each) of macrophages transfected with control or Atg5 siRNA (means and ranges are shown).
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C) Immunoprecipitation and Western blot were performed in HFFs with and without IFNγ (10 U/ml) using an RH strain expressing type II GRA15-HA and as a control RH expressing GRA35-HA. The blots using antibodies against TRAF2 and TRAF6 were made after stripping the first blot. The inputs loaded represent 1% of total lysate prepared for immunoblotting and mass spec (Table EV1). The antibodies against TRAF2 and TRAF6 were obtained from Santa Cruz Biotechnology (Appendix Table S2). Full length blots for this figure can be observed in the source data for this figure.
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B. Hierarchical clustering based on the 755 most significantly differentially expressed genes between adult F4/80hi and CD11bhi cells. Heat map shows z-transformed log2-expression values from red to blue via white.
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C Representative H&E (scale bar 50 µm) and fluorescent images of cl-Caspase 3 staining in B16 WT and XIAPKO tumours. Nuclei were stained with DAPI (blue). Scale bar 100 µm. Quantification thereof (right panel). The average of cl-Caspase 3 positive cells from 5 randomly selected areas of the tumour (5 mice per genotype) was represented. Dots represent individual mice (n=5).
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N-P) Overexpression of ciACT in NBs (dnab-GAL4) reduces EdU index, quantified in (P) (n=12, 10 brain lobes).
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(c,d) (c) Volcano plot and (d) heat map of differentially expressed (FDR < 0.05) mRNA/lincRNA in sperm from Tesa-inj males. Dashed black lines in (c) represent y = 1.3, equivalent to FDR = 0.05, and x = ±0.03, equivalent to FC = 1.23 and 0.81. Black dots represent top candidates, which all have FC > 2 or FC < 0.5, and FDR < 0.05. Teal dots represent non-significant genes.
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Representative IHC staining for ADC (TTF-1) and SCC (KRT5 and ∆Np63) marker expression as well as Usp28 and GFP abundance in KPSCC sh-NTC (n=3) and KPSCC sh-USP28 (n=3) lung tumours. Scale bar =50 µm
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EZH2 inhibitor GSK343 impaired ZNF596 overexpression-reversed and LINC00115 knockdown-inhibited neuro-like sphere formation. 1123 and 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox (2 μg/ml) and/or EZH2 inhibitor GSK343 (1 μM) for 7 days. Data information: data are representative of three independent experiments. Error bars, ± SD. by two-tailed t-test
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Human bronchial epithelial BEAS-2B cells were infected with A/Scotland/20/74 (H3N2) virus (IAV) at MOI=1. After 4 h, cells were treated or not with succinate (Suc; 4 mg/ml) up to 20 h. Human alveolar epithelial A549 cells were infected with the recombinant influenza A/WSN/33 virus expressing a fusion NS1-eGFP protein at MOI= 0.5 for 4 h, then treated with 4 mg/mL of succinate. A549 cells were monitored for 24 h using a BioStation IM-Q device. (d) Single-cell dynamics of the nuclear/cytoplasmic fluorescence ratio. Data are represented as the mean ± SEM of 3 independent experiments with 3 technical replicates each Statistical analysis was performed using the Mann-Whitney test , (*P < 0.05).
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(B) Overrepresented phosphorylation-sequence motifs calculated with MotifX. Phosphorylation sites with a fold change ≥ 2 in setup SR and ≤ 0.5 in setup SR igo1∆igo2∆ were compared to the whole set of phosphorylation sites quantified in setup SR and setup SR igo1∆igo2∆ as background.
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(C) Representative western blot of ORF65 levels at 0, 24 and 48 hours post induction in scr and DLL4 KD TREx cells, GAPDH was used as a loading control and densitometry analysis performed on n=3. Data information: data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (Unpaired Student's t-test). All repeats are biological.
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(e) Representative traces of Ca2+ uptake kinetics in digitonin-permeabilized TbMCU BSF trypanosomes cultured in the absence (−Tet) or presence (+Tet) of tetracycline. The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ca2+ uptake was monitored over time using 1 μM Calcium Green-5N. Ruthenium red (RR, 40 μM, yellow and red tracings), oligomycin (Oligo, 2.5 μg ml−1), EGTA (0.4 μM) and CaCl2 (pulses of 0.4 μM) were added where indicated. (f) Relative Ca2+ uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 (−Tet) (means±s.d., n=3, *(+Tet±RR), **(−Tet±RR), ***(−Tet/+Tet) , P0.001, Student's t-test).
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Top: Oct4-GFP (green) reporter expression (left), and Blimp1-RFP (red) and Oct4-GFP (BROG) reporter expression (right) in the gonads of E13.5 embryos generated by 4N complementation with the indicated ESC lines. Scale bar: 100um. Bottom: percentage of Oct4-GFP reporter-positive PGCs (left) and BROG reporter-positive PGCs (right) measured by FACS analysis. Blue circles, red squares, and green triangles represent the percentage of gonadal PGCs in the first, second, and third independent mutant line, respectively, of the examined locus (L#1-5).
|
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c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).
|
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(A) Confocal micrographs of adult brain of a young (2 d old) wild-type fly. Positive staining for Ref(2) P and ubiquitin is not evident. (B-D) Confocal micrographs of adult brain of an old (8 wk old) wild-type fly. Ref(2) P accumulates in protein aggregates that often colocalize with ubiquitin (arrows). The white rectangle in C indicates the area shown in D.
|
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(A) Total lysates of different mouse organs underwent immunoblotting analysis using anti-Pdia4 and anti-actin antibodies. Data information: Data from 3 experiments are presented as the mean ± SD.
|
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(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).
|
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