EMBO/sd-character-level-ner huggingface dataset:
Figure 4Changes in WT and variant FOXL2 mRNA-expression levels were assessed by real-time RT-PCR (A) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h. The data (mean ± SEM) are from three independent experiments, performed in triplicate. Changes in WT and variant FOXL2 mRNA-expression levels were assessed by western blot analysis (B) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h. The data (mean ± SEM) are from three independent experiments, performed in triplicate. C The mRNA levels of WT (left) and variant FOXL2 (right) were determined in KGN cells by real-time RT-PCR, after transfecting a control miRNA or miR-1236. The data (mean ± SEM) are from three independent experiments, performed in triplicate. D The mRNA levels of WT and the variant FOXL2 in control (left) and miR-1236-/- KGN cells (right) after transfecting siRNAs against AGO mRNAs were determined by allele-specific real-time RT-PCR. The data (mean ± SEM) are from three independent experiments, performed in triplicate. E 293T cells were transfected with an miR-1236 mimic (50 nM) for 24 h, followed by cotransfection with expression vectors encoding FLAG/HA-tagged variants of the indicated human AGOs and pGL3c-CDS-MT for 24 h. The empty p3XFLAG-CMV-10 vector was used as control. Co-immunoprecipitated mRNAs were reverse transcribed, and the cDNA products were used for allele-specific real-time PCR analysis of the FOXL2 variant and GAPDH mRNAs (top). The level of variant FOXL2 mRNA immunoprecipitated using FLAG-tagged AGO proteins was normalized using the level of GAPDH mRNA from the same lysates. The immunoprecipitated-AGO proteins were detected by western blotting (bottom). The data (mean ± SEM) are from three independent experiments. F In vivo association of AGO3-mediated miRISC formation with FOXL2 mRNAs are shown. Following transfection of a control miRNA or the miR-1236-G mutant into KGN cells, AGO3-mediated RISC-associated RNAs were isolated by immunoprecipitation with an anti-AGO3 antibody. IgG was used as a control. The co-immunoprecipitated mRNAs were reverse transcribed using a FOXL2-430-R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele-specific PCR analysis with the FOXL2-279F primer (Appendix Fig. S1), and a representative result (top left) is shown. Quantitative real-time RT-PCR results that examined FOXL2 mRNAs, normalized using the level of GAPDH mRNA (bottom left), are also presented. Western blot analysis of immunoprecipitated AGO3 and inputs are shown in the right panel. The data are presented as the mean ± SEM of two independent experiments. G RNA-seq analysis was performed to determine AGO-expression levels (transcripts per million) from the individual tissues from 20 independent AGCT patients. X-axis represents mRNAs of AGO1 to 4. The box plot represents the minimum value, first quartile, median, third quartile and maximum value of a data set. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots.
a, GFP–NCOA4 (green) co-localizes with endogenous LC3B (red) in chloroquine (CQ)-treated cells.f2a, GFP-NCOA4 (green) co-localizes with endogenous LC3B (red) in chloroquine (CQ)-treated cells. CTRL, control. Scale bar, 20 μm. b, GST pull-down assay of NCOA4-HA-Flag from stable 293T cells using GST-ATG8 proteins. HA immunoblot for NCOA4-HA-Flag. c, Lysates from PA-TU-8988T cells treated with chloroquine or BAF (8 h) were immunoblotted for NCOA4, LC3B and ACTB as a loading control. Asterisk indicates cross-reactive band (see Extended Data Fig. 7d, e). d, PA-TU-8988T autophagosome purification fractions were analysed using antibodies to NCOA4 and LC3B. TAX1BP1, a newly identified autophagy receptor, was included as a positive control. A1, autophagosome fraction; A2, autophagolysosome fraction; L, lysosome fraction; LD, gradient load; M, mitochondrial fraction. e, NCOA4 interaction network from cells expressing NCOA4-HA-Flag or FTH1-HA-Flag (Supplementary Table 9). Black lines (this study) depict directionality of interaction observed with line thickness weighted by weighted and normalized D score (WDN) score (293T data set). Dotted lines indicate data derived from the STRING database. Numbers in parentheses indicate log2(H:L) ratio of NCOA4, FTH1 and FTL from MCF7 Ex. 1 data set (Supplementary Table 4). f, g, Extracts from 293T cells stably expressing the indicated proteins were immunoprecipitated with anti-Flag (Flag-IP) and immunoblotted with the indicated antibodies. Anti-ACTB, loading control. h, Representative confocal images of GFP-NCOA4 (green) and ferritin (red) after no treatment or FAC treatment (24 h). Scale bar, 10 μm.
Mitochondrial dysfunction is a feature of cardiomyocyte senescence and drives TAF in mouse cardiomyocytes in vivo.Figure 6Mito and ETC genes with GSEA analysis: Clustered heatmap showing all genes associated with the "Mitochondrion" GO term in young and old, mouse CMs as observed by the GSEA pre-ranked list enrichment analysis (normalised enrichment score: -1.70; FDR q-value < 0.05). Alongside this is a column clustered heatmap displaying a list of genes from the electron transport chain (ETC) GO ontology. In both instances, genes are by column and samples by row with the colour intensity representing column Z-score, where red indicates highly and blue lowly expressed. Real-time PCR gene expression analysis of MAO-A, MnSOD and catalase in isolated mouse CMs from young (3 months) and old (20 months) mice. Mean % of 4-HNE- (top) or 8oxodG- (bottom) positive CMs from 3 month (young) or 30 month (old) aged mice. Data are mean ± SEM of n=4 per age group. 100 CMs were quantified per age group. Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in wild-type (control) compared to MAO-A transgenic mice with or without drinking water supplemented with 1.5g/kg/day NAC from the age of 4 to 24 weeks Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group. Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in MnSOD+/+ vs MnSOD-/+, Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group. Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in Catalase+/+ vs. Catalase-/-, Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group. Mean % of TAF-positive nuclei (left graphs) or mean % of TAF (right graphs) in WT vs. POLG double mutant mice. Data are mean ±S.E.M of n=3-4 per group. >100 CMs were quantified per age group. Schematic depicting isolated mouse adult CMs isolated from 4 animals were treated with or without 100nM rotenone either in the presence of 5mM NAC or vehicle control (pre-treated for 30 minutes before rotenone treatment), for 24 h before fixation. Mean number of TAF (top graph) and mean % of TAF-positive nuclei (bottom graph). Data are mean ± SEM from 4 separate CM cultures isolated from 3 month-old mice. 50 CMs were quantified per condition.
Extensive functional heterogeneity within tumor-infiltrating CTLs in vivo(A-B) CTLs exhibit distinct capacities to trigger calcium responses in contacted tumor cells.Figure 5(A-B) CTLs exhibit distinct capacities to trigger calcium responses in contacted tumor cells. Mice with established B cell lymphoma expressing OVA and the Twitch-2B calcium indicator were adoptively transferred with GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. (A) Left. Two-photon time-lapse image and corresponding scheme showing representative CTLs triggering either limited (upper images) or numerous (lower images) calcium signals in 5 contacted targets. Red arrows mark tumor cells with elevated calcium. Scale bar, 5 µm. Right. The periods of contact for the indicated targets are shown as white boxes and periods of calcium elevation are shown in magenta. (B) Total number of distinct calcium fluxes triggered by individual CTLs in all contacted tumor cells during an imaging period of one hour. For a rigorous comparison, only CTLs which activity could be monitored for at least 1 h were included in the analysis. Data are representative of 4 independent experiments. CTLs exhibit extensive differences in lytic activity in vivo. Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct-GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. The frequency of CTLs engaged in lytic activity (induction of apoptosis in at least one contacted tumor cell) is shown (C) together with the frequency of lytic contacts within all CTL-tumor cell contacts recorded (D). CTLs exhibit extensive differences in lytic activity in vivo. Mice with established B cell lymphoma expressing OVA and the caspase 3 reporter were adoptively transferred with LifeAct-GFP-expressing OT-I CTLs. Intravital imaging of the bone marrow was performed two days after T cell transfer. (E) Representative two-photon time-lapse images showing a representative CTL with high lytic capacity (killing 4 targets) and a CTL with no lytic activity for up to 4 hours. Blue arrows mark apoptotic tumor cells. Live and apoptotic tumors appear in grey and blue, respectively. CTLs appear in green. Scale bar, 10 µm. Representative of 3 independent experiments.
Figure 6(A) Heatmap of differentially regulated genes in skin lesions from LL37-injected mice administrated with RAPA or vehicle determined by RNA-sequencing (n = 3 independent biological samples for each group). Blue color denotes low FPKM expression; red, high FPKM expression. (B) Venn diagram showing the overlap genes between the two comparisons. LL37 vs control, LL37-injected mice administrated with vehicle vs control mice administrated with vehicle; LL37 vs LL37+RAPA, LL37-injected mice administrated with vehicle vs LL37-injected mice administrated with RAPA. (C) Top-ranked enriched KEGG terms in genes that were differentially regulated respectively in the two comparisons (LL37 vs control, and LL37 vs LL37+RAPA) revealed by GSEA analysis. NF-κB signaling pathway was highlighted in red box. (D) GSEA on RNA-sequencing data from the two comparisons both shows enrichment for NF-κB signaling pathway in LL37 group. Significance was calculated by permutation test. (E) Immunostaining of phospho-p65 (p-p65) in skin sections. Bottom right panels, magnified images of dotted line areas. Epi, epidermis. Der, dermis. Scale bar: 50 μm. (F) Quantification of relative fluorescence intensity for p-p65 in epidermis (n=6). (G) The mRNA expression levels of NF-κB family of transcription factors (Nfκb1, Nfκb1, Rela and Relb) in mice skin lesions (n=6 for each group). (H) GSEA on RNA-sequencing data shows enrichment for NF-κB signaling pathway in rosacea patients. Significance was calculated by permutation test. (I) IHC of p65 on skin sections from HS and rosacea. Scale bar, 50 μm. (J) Percentage of nuclear p65 positive cells in the epidermis from HS (n = 8) and rosacea (n = 13). All results are representative of at least 3 independent experiments. Data represent the mean ± SEM. **P < 0.01. 1-way ANOVA with Bonferroni's post hoc test (F and G) or two-tailed unpaired Student's t-test (J) was used.
Figure 4A. Effects of GOLPH3 KD or OE on glycosphingolipid synthetic flux as assessed by [3H]-sphingosine pulse (2 hr) and chase (indicated times), lipid extraction and HPTLC separation and radioactive counting (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). B. Effects of GOLPH3 KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on sphingolipid levels as assessed by LC/MS of HeLa cells (bottom) (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs. C. Effects of GOLPH3 KD or OE on Gb3 as assessed by MALDI-MS after lipid extraction. Profiles are representative of 3 independent experiments. Untargeted lipidomics data from this study are available at Metabolomics Workbench database with the following ID: ST001674. D. Histograms reporting the percentage of ShtxB or ChTxB positive cells in each condition as in Fig. 1F (data are means ± SD of at least 3 independent experiments; *p <0.05, ***p <0.001 [Student's t-test]). E. Effects of GOLPH3 KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (top) and PHFs (middle). Effects of LCS KD or OE on ceramide levels as assessed by LC/MS of HeLa cells (bottom). The dotted black box highlights the effects on C16:0 Cer (data are means ± SD of at least 3 independent experiments; *p <0.05, **p <0.01, ***p <0.001 [Student's t-test]). Targeted lipidomics data from this study are available at Metabolomics Workbench database with the following IDs: ST001673 for HeLa cells, and ST001672 for PHFs.
Figure 3A. Co-immunoprecipitation of ectopic IN1 using C160-HA as bait, from yeast protein extracts expressing WT or mutated IN1-Strep (K617A, S621A, or L622A) from the GAL1 promoter in the presence of galactose. Expected sizes are 160 kDa for C160-HA and 100 kDa for IN1-Strep (WT and mutants). B. Quantitative ChIP analysis of HA-IN1 enrichment at Pol III-transcribed genes. Immunoprecipitated DNA from yeast cells producing ectopic IN1 is expressed as a value relative to that of the input. Pol III transcribed-genes: tDNA-Ile and tDNA-Leu families (16 and 22 genes, respectively) and the unique SCR1 gene. GAL1 ORF serves as a control. Data represent means ± SD (n ≥ 3). C. Fold enrichment of WT HA-IN1 over input at all the genes where HA-IN1 has been detected by ChIP-seq analysis. Each dot represents a gene. Grey, tDNAs ; Red, other Pol III-transcribed genes ; Blue, Pol II-transcribed genes. The dashed line indicates the separation between Pol II and Pol III-transcribed genes. D. WT and mutant IN1 association with all Pol III-transcribed genes. Values obtained from ChIP seq analysis have been normalized in log2 RPKM (reads per kilobase per million mapped reads). Control is anti-HA immunoprecipitation of chromatin extracts in cells expressing IN1-Strep. Immunopurified DNA from 3-4 independent experiments was combined in one replicate. Data are presented as boxplots showing the median, the first quartile, and the third quartile. Error bars show minimum and maximum values. E. Genome-wide occupancy profiles (top) and heatmaps (bottom) of WT and HA-IN1 mutants over a 1kb window upstream and downstream of all tDNAs. Pearson correlation to WT values corresponding to Fig EV2B are indicated. F. WT and mutant IN1 association to all Pol-II transcribed genes respectively. Control, replicates and data presentation, as described for panel D. Regions used for calculation are gene bodies. G. Recruitment of IN1 along the SCR1 gene locus. Top. ChIP‐qPCR analysis as described for panel Fig 3B. Data represent means ± SD (n = 3). Bottom. Schematic representation of the SCR1 locus with DNA amplicon positions. TATA, A and B boxes and terminator of transcription (T) are indicated.
Figure 4A) Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the TA and triceps muscles of the Dup18-30 mice. A representative image is shown. Scale bars, 100 μm. B) TA, triceps and diaphragm muscle architecture was analyzed by H&E staining. Scale bars, 50 μm. C) Central nuclei were quantified in the TA, triceps and diaphragm muscles. Dup18-30 untreated, n = 4-5; Dup18-30 treated n = 6-9. All data is represented as means ± S.D. Statistical analyses were performed with Student's t-test. *** P< 0.001, **** P< 0.0001.
Figure 1A. RT-PCR analysis of the expression of β-actin, Trpv1, Trpv2, Trpv3 and Trpv4 using mouse differentiated brown adipocytes after 29 (upper) and 35 (lower) thermal cycles with (RT (+)) and without (RT (-)) reverse transcription (RT). Control (Ct.) lanes indicate the results with each plasmid DNA as a template. B. Results of real-time RT-PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse differentiated brown adipocytes. Expression levels of mRNA were normalized to that of the ribosomal protein gene (36B4), a housekeeping gene un-affected by adipogenesis. Data are presented as mean ± SEM, n = 6. C. Results of real-time PCR analysis of Trpv1, Trpv2, Trpv3 and Trpv4 expression using mouse interscapular brown adipose tissue (iBAT). mRNA expression levels were normalized to that of 36B4. Data are presented as mean ± SEM, n = 5. D. Western blot results of TRPV2 and tubulin from WT and TRPV2KO brown adipocytes. Upper bands in the TRPV2 blots likely indicate glycosylated forms. E. Averaged traces of [Ca2+]i changes in response to 500 µmol/L 2APB in differentiated brown adipocytes from WT (black) and TRPV2KO (red) mice. One µmol/L NE was used to confirm differentiation. Five µmol/L ionomycin was used to confirm cell viability. Ratio values correspond to the real [Ca2+]i of differentiated mousebrown adipocytes. Data are presented as mean ± SEM, n = 149 of WT cells, and n = 112 of TRPV2KO brown adipocytes; ** P < 0.01. Unpaired Student's t-test. F. A representative trace of whole-cell current activated by 3 mmol/L of 2APB in the presence or absence of 10 µmol/L SKF in mouse differentiated brown adipocyte. The left inset indicates a voltage ramp-pulse protocol. The right inset indicates the current-voltage curves of basal current (a, black), current in the presence of 2APB+SKF (b, blue) and current in the presence of 2APB alone (c, red) at the time points of a, b and c, respectively.G. Comparison of the mean densities of basal currents (Base, black), currents in the presence of 2APB alone (red) and currents in the presence of 2APB+SKF (blue) at -60 mV and +100 mV in mouse differentiated brown adipocytes. Data are presented as mean ± SEM, n = 10; ** P < 0.01 vs. Base; ##P < 0.01 vs. 2APB alone. One-way ANOVA followed by 2-tailed t-test with Bonferroni correction.
Figure 7(A) Western blotting to examine the expression levels of H2B and H4 in Cntl, ALYREF and ARS2 KD cells. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot (B) Growth curve of Cntl and ALYREF KD cells. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01. (C) Cell cycle analysis of Cntl, ALYREF and ARS2 KD cells. Quantifications of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle are shown in the upper right corner of each histogram. (D) Model for ALYREF promoting histone mRNA 3'-end processing and nuclear export.
Figure 3A, Immunoblot analysis of protein lysates from HeLa cells treated with 250 μM NaAsO2, EBSS, 250 nM Torin-1, 15 mM NAC or a combination of these drugs with NAC for 1 h and run under non-reducing condition. B, Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one-way ANOVA (*)p<0.05, (***)p<0.001, and (****)p<0.0001 from two independent experiments. 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. D, Immunoblot analysis of protein lysates from ARPE-19 cells overexpressing TFEB-FLAG or TFE3-MYC, treated with 250 nM Torin-1 for 1 h and run under non-reducing condition. E, Quantification of immunoblot data shown in (D). Data are presented as mean ± SD using two-tailed t-test (*)p<0.05 from three independent experiments. F, Immunoblot analysis of protein lysates from ARPE-19 cells overexpressing TFEB-FLAG-WT, TFEB-FLAG-S211A, TFE3-MYC-WT or TFE3-MYC-S321A and run under non-reducing condition. G, Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using two-tailed t-test (*)p<0.05 and (**)p< 0.01 from three independent experiments.
Figure 7A) Immunofluorescence against NeuF (green) and MHC (red) revealing reconstructed muscle innervation (asterisks). B) Superimposed image in (a) over LacZ stained image. C) Syn and BTX immunolabelled sections showing neuromuscular junction formation (arrowheads), inset enlarged view of dashed box. D) Electrophysiological analysis scoring muscle strength recovery performed in vivo on ablated TA (control) and wet-spun implanted contralateral TA (graft) (n=3), statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *55Hz=0,016830182; *75Hz=0,012686264; *100Hz=0,009143348; *150Hz=0,009327048). E) Rate of coupled versus uncoupled BTX-Syn signals in reconstructed TA NMJ.
Knock-in cell lines A Scheme of the knock-in alleles of the SOX1-BRA-Reporter-OCT4-HALO-SOX2-SNAP (SBROS) cell line.Figure 1Scheme of the knock-in alleles of the SOX1-BRA-Reporter-OCT4-HALO-SOX2-SNAP (SBROS) cell line. Red hexagons: STOP codons. Yellow triangles: loxP sites. Example images showing the localization of SOX2-SNAP and OCT4-HALO by fluorescence microscopy of SBROS cells. Scale bar: 50µm. Correlation between OCT4-HALO and total OCT4 levels determined by immunofluorescence and HALO labelling. R is Pearson's correlation coefficient. Distributions of OCT4 (n=2682), SOX2 (n=1236) and NANOG (n=2416) levels in WT E14 and SBROS cell lines as determined by quantitative immunofluorescence. Dashed lines: mean protein levels; Dotted lines: median protein levels. Protein half-lives of OCT4-HALO and SOX2-SNAP in SBROS cells. Whiskers: minimum and maximum values; Box: lower and upper quartiles; Solid line: median. Black dots: outliers. Cell cycle duration of E14 WT (n=6) and SBROS (n=8) cells. Error bars: SE. p-value: two-sided t-test with unequal variance.
CHK1-inhibition kills human cord-blood derived CD34+ HSPCs in a BCL2 regulated manner.Figure 3MACS-purified CD34+ human cord-blood derived HSPC were treated with PF-477736 or CHIR-124 ± the caspase-inhibitor QVD (50 μM). Cell death was assessed using AnnexinV/7-AAD staining and flow cytometry. CD34+ cells were transduced with a BCL2-encoding lentivirus or a control virus, along with IRES-GFP with a MOI of 10 for two consecutive days, followed by CHK1i application for 48 h. Survival was assessed using Nicoletti staining and flow cytometry. Percentage of living GFP+ transduced human CD34+ HSPCs, cultured in the absence or presence of CHK1i for 48 hours. Colony formation potential of human CD34+ HSPCs transduced with empty control or BCL2 encoding virus. Colonies were counted 10 days post seeding of 1000 CD34+ cells per plate/sample. "Myeloid colonies" include granulocyte/monocyte, monocyte, granulocyte and GEMM (granulocyte, erythroid, monocyte/macrophage and megakaryocyte) colonies.