EMBO/sd-character-level-ner huggingface dataset:
Genome‐wide screen for starvation‐induced autophagy.Figure 1(A) Cellomics images (top) of GFP-LC3‐HEK cells in full medium (FM) or after 2 h in starvation medium (ES) with leupeptin (EL). Scale bars=40 μm. Optimised automated analysis (bottom left) programme detecting hoechst‐labelled nuclei (purple line), cell outline (red line) and identifies GFP-LC3 spots (red spots) in FM and EL. Enlarged GFP-LC3 images in FM and EL (bottom right). Scale bars=10 μm. (B) SCPO, STIPO and STAPO after indicated siRNA treatment of GFP-LC3‐HEK cells incubated as in (A). Error bars represent s.e.m. Significance was determined using a two‐tailed paired t‐test compared with RISCfree (RF) in EL: SCPO siULK1, ***P=0.0002; siATG7, **P=0.0078; siNRBP2, **P=0.0088. STIPO siULK1, ***P0.0001; siATG7, **P=0.0060; siNRBP2, **P=0.0041. STAPO siULK1, ***P0.0001; siATG7, **P=0.0037; siNRBP2, **P=0.0028. The experiments were performed: RISCfree (n=5), siULK1 (n=5), siATG7 (n=3), siNRBP2 (n=3). (C) Overview of screening strategy. (D) Scatter plot of median SCPO Z‐scores from siRNA pools in the primary screen that met the minimum object count (OC) cutoff. Dashed lines represent approximate cutoff for 500 best increasers and decreasers. RP of known autophagy genes (ATG4C, ATG14, RB1CC1, ATG9A, ATG7) are given in Supplementary Table S2; AMBRA1 (FLJ20294), TBC1D25 (OATL1) and SH3GLB1 (BIF‐1) in Supplementary Table S1; LAMTOR2 has a RP=708.7 and RUBICON has a RP=1126.
Figure 1Protein levels of E2F7 and E2F8 during cell cycle progression. HeLa cells were synchronized by a double thymidine block and released into fresh medium. Cells were harvested at the indicated time points and an asynchronous (AS) condition was used as control. Protein levels were measured by immunoblotting and cell cycle progression was determined by flow-cytometry (shown in Fig EV 1A). The asterisk indicates the E2F7-specific band. Decreased stability of E2F7 and E2F8 in nocodazole-arrested cells. HeLa cells were either treated with DMSO or nocodazole (50 ng/ml) for 16 hours. Cells were harvested and lysed for immunoblotting. Protein expression of cyclin B1 was used as a marker for G2 or M, and γ-tubulin was used as loading control. Selective Cullin-RING inhibitor MLN4924 rescued the degradation of E2F7/8 under nocodazole-arrested condition. HeLa cells were treated with DMSO, nocodazole or nocodazole plus MLN4924 (0.1 μM) for 16 hours. Cells were harvested and lysed for immunoblotting. cyclin B1 expression was used as a marker for G2 or M cell cycle progression, and γ-tubulin was used as loading control. Increased half-life of E2F7/8 by MLN4924 treatment. HeLa cells were either treated with cycloheximide (CHX, 50 ug/ml) with or without MLN4924 (0.1 μM). Protein levels of E2F7 and E2F8 were determined by immunoblotting (left panel). Asterisk indicates the E2F7-specific band. cyclin B1 and cyclin A2 expressions were used as a marker for G2 or M cell cycle progression, γ-tubulin was used as loading control. Quantifications (right panels) were performed based on two independent experiments. Bar and error bars represent mean ± SEM.
Suppression of Piwi1 expression reduces normal metabolic functions.Figure 4A Gene ontology enrichments for biological processes in the downregulated genes in the fat body following dsPiwi1 treatment. The heat map shows the gene ratio percentage and the number of differentially expressed genes for a given GO term/the number of all the locust genes for a given GO term. B Representative vertical images of dsPiwi1-treated locusts and dsGFP-treated locusts on the fourth day of the fourth instar. C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). The scale bars represent 50 µm. D Quantification of lipid droplet staining in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). E Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts. E, F Measurements of glycogen (n=5 biological replicates) and TAGs (n=10 biological replicates) in fat bodies. Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.
Ac (a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.Figure 2(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+. (b) In vitro deacetylation of recombinant SUMO2WT, SUMO2K11Ac, SUMO2K33Ac and SUMO2K35Ac. Immunoblotting was performed with antibodies as indicated. (c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac. (d) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 (1.5 μM). (e) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1, followed by substrate dependent in vitro SUMOylation of p53. (f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.
Single cell transcriptome atlas for human neural retina.Figure 1(A, B) t-SNE visualization of 20,009 human retinal cells colored by (A) annotation of 18 transcriptionally distinct clusters (C0-C17) and (B) their distribution in 3 donor retina samples.  C) Feature expression heatmap showing expression patterns of major retinal class markers across 16 retinal cell clusters. The size of each circle depicts the percentage of cells expressing the marker within the cluster. Brown colour indicates ≥10 nTrans (number of transcripts). D) t-SNE plots showing expression of a set of selected marker genes for major retinal classes. E) Correlation matrix for the identified 18 clusters. The upper triangle depicts the z-value for correlation and the lower triangle depicts the correlation coefficient for gene expression in clusters. F) Heatmap of differentially expressed genes used to classify cell types for each cluster compared to all other clusters for the 18 retinal cell clusters. The rows correspond to top 10 genes most selectively upregulated in individual clusters (p< 0.01, Benjamini-Hochberg correction) and the columns show individual cells ordered by cluster (C0-C17).
Anaerobic cultivation of the different strains under growth arrest.Figure 2A) Time course of biomass concentration. B) Time course of glucose concentration. Data information: The means for n = 3 biologically independent samples are shown. The error bars represent ± SD.. Note: although no nitrogen source was present in the medium, some minor growth (especially of the control and wild type strains) remained (A), which is a known phenomenon within the first hours of cultivation after nitrogen depletion C) Specific glucose (Glc) uptake rate and specific productivity for ethanol (Eth), acetate (Ace), formate (For), lactate (Lac), and succinate (Suc). Data information: The reaction rates in C were calculated from the beginning of cultivation until the last sampling time point where glucose was still present in the medium. The means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD.. D) Yield of Eth, Ace, For, Lac, and Suc. Data information: The means and individual data for n = 3 biologically independent samples are shown. The error bars represent ± SD..
STAMP2 affects ATF4 expression in PCa cells.Figure 6A LNCaP cells were transfected with either control or STAMP2-specific siRNA in the presence of 10−8 M R1881. RNA was isolated, and qPCR was used to determine ATF4mRNA levels. Student's t-test was used to analyze the statistical significance, n = 3. *P = 0.002. Error bars indicate SD. B Cell lysates from (A) were subjected to Western blot analysis. C, D ATF4 expression in LNCaP (C) and 22Rv1 (D) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis. C, D ATF4 expression in LNCaP (C) and 22Rv1 (D) cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by Western blot analysis. E ATF4 expression in xenografted tumors of 22Rv1 cells stably expressing control shRNA or shRNA against STAMP2 was analyzed by qPCR. Student's t-test was used to analyze the statistical significance, n = 4. *P = 0.022; **P = 0.015; ***P = 0.044. Error bars indicate SEM. F CWR22 xenografts were grown in nude mice, and tumors were collected at different times after castration. The association between ATF4 and STAMP2 in the tumor samples was determined by Western blot analysis. G The MSKCC Prostate Oncogenome cDNA microarray dataset was obtained from the cBio Cancer Genomics Portal. Analysis was performed as described in Materials and Methods. The expression levels of STAMP2 and ATF4, as well as two ATF4 target genes (ASNS and SLC7A11), are presented.
Figure 5A: SPC105-GBD cells expressing Bub1-GFP were grown at 30°C and filmed every 4 minutes at 30°C by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm. B: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures. C: cells with the indicated genotypes were grown at 25°C and then shifted at 34°C (t=0). Every hour after the temperature shift the same number of cells was plated on YEPD for each strain and incubated at 25°C to determine the number of colony-forming units. Percentages of viable cells have been calculated for each strain relative to t=0. Error bars: SD. N=3. D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C. Cells were collected at the indicated time points for FACS analysis of DNA contents.
Figure 3SENP3 knockdown abolishes the DFP-induced increase in LC3-II in HeLa cells. Nsi or SENP3i (I) was transfected into HeLa cells. Two days post-transfection the cells were treated with DFP for a further 24 h. Whole cell lysate samples were blotted as indicated (Nsi, non-specific siRNA; SENP3i, SENP3 siRNA; siRNA concentration, 20nM; DFP, 1mM for 24 h; values normalized to the control value; n=3, biological replicates; ***, p<0.001; N.S., not significant, p>0.05; paired t-test). Replacement of endogenous SENP3 with GFP-SENP3R WT, but not GFP-SENP3R C532A, restores DFP-induced LC3-II levels. GFP-SENP3R WT or SENP3R C532A was expressed in HeLa cells after knockdown of endogenous SENP3 using SENP3i (II). The cells were treated with DFP (1mM for 24 h) in the presence of CQ (50μM; for final 12 h of DFP treatment). Whole cell lysate samples were blotted as indicated. Establishment of HeLa cells stably expressing mito-Keima (Live image of cells grown in a T75 flask; Scale bar, 100 μm). Nsi or SENP3i (II) was transfected into HeLa cells with stable expression of mito-Keima grown on 6-well plates. Two days post-transfection the cells were treated with DFP (1mM; 24 h). Cells transfected with Nsi and treated with CQ (50μM; 24 h) in the absence or presence of DFP (1mM; 24 h) were included as a negative control for mitophagy induction. The images show overlaid fluorescence and transmitted light images of unfixed, live cells obtained using an EVOS-fl inverted LED fluorescence microscope (Red: mitophagic puncta; Scale bar, 100 μm). Histogram shows the percentages of mitophagic cells under 6 different conditions (n=3, biological replicates; ****, p<0.0001; Ordinary one-way ANOVA followed by Tukey's multiple comparisons test). SENP3 knockdown abolishes DFP-induced mitophagic autolysosomes. Nsi or SENP3i (I) together with mito-pHfluorin were transfected into HeLa cells in the absence or presence of DFP (24 h), and were analyzed using the mito-pHfluorin construct 72 h post-transfection (Red: mCherry-A or puncta indicating occurrences of mitophagy marked by white arrows; Green: SEP-A; Yellow: mitochondria labelled by mCherry-SEP-A; Magenta: SENP3; Scale bar, 10 μm; n=28 cells per condition from two individual experiments; **** p<0.0001; unpaired t-test).
Figure 2(A) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K/O cells after expression of HA-tagged PI4K2A or its kinase-dead mutant. Means ± S.E.M. are shown from three experiments performed in triplicates. (B) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild-type cells. (C) Distribution of GFP-Rab7 in parental (left panels) and two clones of PI4K2A K/O cells. Representative pictures show tubulation in the K/O cells reminiscent of those seen in wild-type cells expressing the GTP-locked Rab7Q67L mutant. Scale bars are 10 µm.
Figure 4A. EPC2-hTERT-EGFR-p53R175H cells grown in OTC stained for RCP. Arrow indicates invasive region. (scale bar = 100 μm). B. Transwell Boyden Chamber invasion assay of WT and mutant p53 cells expressing non-targeting control (NT) and Rab11-FIP1 shRNAs (sh1 and sh2) (n=9 technical replicates from 3 different passages). Error bars represent ±S.E.M. n.s. WT p53 NT vs sh1, ***p=0.0005 WT p53 NT vs sh2, n.s. Mutant p53 NT vs sh1,** p=0.0026 Mutant p53 NT vs sh2, (One-way Anova) *p=0.0173 WT p53 NT vs Mutant p53 NT, (unpaired t-test). C-D. WT and mutant p53 cells expressing non-target control and Rab11-FIP1 shRNAs were grown in OTC. C. H&E staining of OTC with WT p53 (top) and mutant p53 (bottom) with invasive regions. Scale bar = 50 μm. D. Graph shows relative invasive area from OTC of WT p53 non-target control and Rab11-FIP1 shRNAs. n=5 technical replicates in WT p53 NT, n=6 technical replicates in WT p53 sh1, n=6 technical replicates in WT p53 sh2. Error bars represent ±S.E.M. **** p<0.0001 WT p53 NT vs sh1, n.s. WT p53 NT vs sh2(One-way Anova).
Figure 5A. Confocal microscopy images of FANCD2 immunofluorescence staining in control (UVA) or TMP-UVA-treated (2 µM TMP, 2 h followed by 5 s irradiation) WT and PRIMPOL KO cells. Nuclear DNA is counterstained with DAPI. Scale bar, 25 µm. B. Dot plot (left panel) shows the distribution and mean (horizontal red line) of FANCD2 foci number per cell in each condition. Data pooled from three replicates are represented (n ≥100 cells per condition). Statistical analysis was conducted with Kruskal-Wallis test and Dunn´s post-test. p-values of individual comparisons are indicated. Histograms (right panel) show the average foci number and SD of three assays in each case. Circle dots in each column represent the values of individual replicates. Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated. C. Metaphase spreads stained with Leishman, obtained from control (-) or TMP-UVA-treated cells (2 µM TMP, 2 h followed by 5 s irradiation). Scale bar, 10 µm. Representative images of chromosomal breaks and fusions are shown at higher magnification. D. Histograms show the number of chromosomal aberrations per metaphase (average and SEM) in each condition. Data taken from three replicates are combined (50 metaphases per replicate). Statistical analysis was conducted with one-way ANOVA followed by Bonferroni post-test. p-values of individual comparisons are indicated.
Figure 6(A) Structure of the spindle upon NuMA and Eg5 inhibition. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline (Dox), 500 μM IAA and monastrol. Green, red, and blue represent NuMA (mClover), α-tubulin, and DNA, respectively. Z-projections of 10 sections, every 0.3 μm. Scale bar, 5 μm. (B) Frequency of spindle structure patterns upon NuMA depletion and Eg5 inhibition in (A). Values are mean percentages ± SD. from three independent experiments (N≧22 spindles in each experiment). ( (C) The localization of Eg5 in the spindle upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml doxycycline Dox and 500 μM IAA. Gray, green, red, and blue represent α-tubulin, NuMA (mClover), Eg5 and DNA, respectively. Z-projections of 5 sections, every 0.3 μm. Scale bar, 5 μm. (D) Time-lapse observation of the establishment of spindle bipolarity upon NuMA depletion. HCT116 TetOsTIR1 NuMA-mAID-mClover-FLAG cells with 2-centrosomes were treated with 1 μg/ml Dox and 500 μM IAA and observed with a 40× objective. Red and green represent SiR-tubulin and NuMA, respectively. Z-projections of 14 sections, every 2 μm. Scale bar, 10 μm. Time zero corresponds to NEBD. (E) The time required for the initial establishment of spindle bipolarity in (D). Line and error bars represent the mean and SD. (N≧60 cells from two independent experiments). The Mann-Whitney U test (two-tailed) was used to obtain a P value.
Figure 6(A) Sequences of the neck region of human and long and short isoform of mouse DNGR-1 aligned in Clustal X software. Parts of the neck corresponding to the block deletions are depicted as green bars. (B) and (C) Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS-PAGE and Western blot under reducing or non reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +. (D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.
a) Increased Beclin1–PI(3)KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions.figf6(a) Increased Beclin1-PI(3) KC3 interaction by UVRAG and continued interaction of UVRAG with Beclin1 on stress conditions. HCT116.vector, HCT116.UVRAG and HCT116.UVRAGΔCCD cells were used for immunoprecipitation with anti-PI(3) KC3 followed by immunoblotting with anti-Beclin1. WCLs were used for immunoblotting with anti-Beclin1 and anti-PI(3) KC3. HCT116.UVRAG cells expressing Flag-tagged UVRAG were incubated under nutrient-rich conditions (lanes 1 and 2), starvation for 4 h (lane 3), or 2 μM rapamycin treatment for 2 h (lane 4). WCLs were then used for immunoprecipitation with anti-Beclin1 (lanes 2, 3 and 4) or preimmune antibody (lane 1) followed by immunoblotting with anti-Flag. The raw data for this experiment are presented in the Supplementary Information, Fig. S5. (b) Increased PI(3) KC3 activity by UVRAG. At 48 h posttransfection with Flag-tagged PI(3) KC3 with Beclin1 (lane 1), with Beclin1 + UVRAG (lane 2) or with Beclin1 + UVRAGΔCCD vector (lane 3), WCLs of 293T cells were used for immunoprecipitation with anti-Flag followed by an assay for PI(3) KC3 lipid kinase activity, as described in the Methods. At 16 h posttransfection with p40(phox) PX-EGFP fusion, HCT116.vector and HCT116.UVRAG cells were detected using an inverted fluorescence microscope. p40(phox) PX-EGFP-positive vesicles were quantified as the mean ± s.d. of combined results from three independent experiments. The scale bars represent 10 μm. (c) Suppression of autophagy by γHV68 vBcl-2. At 18 h posttransfection with GFP-LC3 vector, NIH3T3.vector, NIH3T3.vBcl-2 and NIH3T3.vBcl-2AAA, cells were incubated under normal or starvation conditions for 4 h. Subsequently, GFP-LC3 was detected using an inverted fluorescence microscope and autophagy was quantified as mean ± s.d. of combined results from three independent experiments.
Figure 2U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm. U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm. (C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm. (D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules. (E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).
Figure 4A S-HAD treatment induces mitochondrial ROS. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 24 h), rotenone or vehicle control as indicated. Mitochondrial ROS was measured by MitoSox staining (4 µM, 15 min) and quantitative confocal imaging. Error bars indicate SD. n=3, asterisk indicates p<0.05 (Student's T-test), n.s., not significantly different. B Quenching mitochondrial ROS reduces cyclin C-Bax interaction. Wild type and Ccnc-/- MEF cultures were treated with S-HAD (10 µM, 16 h) and 100 nM mitochondrial-specific reactive oxygen scavenger MitoTempol. Western blot analysis was conducted as described in Fig. 1. Molecular weight markers (kDa) are indicated on the left. C Insulin treatment stimulates S-HAD induced Bax activation. Wild type and Ccnc-/- MEF cultures were grown with or without insulin (10 µg/ml) then treated with S-HAD (10 µM, 2 h). Mitochondrial and cytosolic fractions were prepared and probed for the indicated proteins. Atp5A and ß-actin levels control for mitochondrial and cytosolic loading, respectively. The two blots were prepared, processed and developed identically. Molecular weight markers (kDa) are indicated on the left. D, E Model for Bax-mitochondrial association before (D) and after (E) PCD-inducing stress. In unstressed cells, Bax continuously samples the MOM but does not insert into the MOM and is removed by pro-survival proteins (Bcl-2, Bcl-XL) in a process termed retrotranslocation. In response to stress, cyclin C associates with Bax stabilizing its interaction with the MOM in a fission complex dependent manner. Increased dwell time makes Bax more efficiently targeted by BH-3 only proteins activated by stress. BH-3 only protein binding induces conformational changes promoting Bax MOM insertion, homodimer formation and oligomerization ultimately leading to PCD.