EMBO/soda-nel huggingface dataset:
Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. (2012)
Figure 5

Poliovirus entry, translation, and RNA replication are unaffected by treatment with inhibitors of vesicle acidification.

(A) H1-HeLa cells were infected with PV at an MOI of 50 pfu/cell. Cells were pulsed at the indicated h.p.i with 35S-labeled methionine for 1 h then lysed. Lysates were run on SDS-PAGE. Expected viral proteins are labeled according to recognized banding patterns. (B) Triplicate plates of H1-Hela cells were infected with PV at an MOI of 0.1 pfu/cell, virus RNA and host GAPDH RNA were measured by qRT-PCR. Virus RNA levels were normalized to GAPDH levels using the delta-Ct method. NH4Cl treatment was as described in Figure 4, and Guanidine HCl (2 mM) was added to the media at the time of infection. The data shown are pooled from three replicate experiments, and the titer of cell-associated virus collected at 6h.p.i. from each replicate was determined by plaque assay. (C) Triplicate plates of 293T cells were treated with 20 mM 3-MA for 2 hours prior to infection, and kept under treatment throughout infection. PV infections were done at an MOI of 0.1 pfu/cell. RNA levels and virus titers were analyzed as in (B). ** p<0.01, *** p<0.0001.
Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. (2012)
Figure 6

Virion maturation requires acidic compartments.

(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with NH4Cl. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 or 6 h.p.i., and lysates were then separated on a 15–30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. Representative gradients from three independent experiments are shown. (B) The three fractions representing the 150S peak in each experiment were pooled for plaque assay analysis. Data shown are the averages from three independent experiments. (C) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE, and the 35S-Methionine labeled bands were visualized. The bands are labeled according to expected relative migration pattern, and VP2 is identified by its absence in the 75S peak. The ratio of VP0 to VP3 bands was analyzed from four independent experiments and plotted in arbitrary units. * p<0.05, ** p<0.01, *** p<0.0001.
Intracellular vesicle acidification promotes maturation of infectious poliovirus particles. (2012)
Figure 7

Proton pump inhibition also affects virion maturation.

(A) H1-Hela cells were infected at an MOI of 50 pfu/cell, and half of the samples were treated with bafilomycin A1 as described in Figure 4. Cells were labeled with 35S-Methionine from 3 h.p.i. until collection at 5 h.p.i., and lysates were then separated on a 15–30% sucrose gradients. Fractions were then collected and the counts per minute (CPM) were analyzed for each fraction. (B) Three fractions representing the 150S and 75S peaks were pooled and run on SDS-PAGE as in Figure 6. The ratio of VP0 to VP3 bands was analyzed and plotted in arbitrary units. The titer of the infectious virus in the pooled 150S fractions was determined by plaque assay. * p<0.05.
Interactions between lineage-associated transcription factors 
govern haematopoietic progenitor states (2020)
Figure 5

Figure 5 - Genome-wide annotation of chromatin states highlights putative primary transcription factor targets.

A Dice scores summarising overlaps among peaks identified for 14 ChIP experiments in Hoxb8-FL cells.

B Comparison of genes identified as differentially expressed following TF loss with mapped ChIP peaks in a corresponding experiment. Enrichment over a random value and the p-value (based on the hypergeometric test) are shown (bars corresponding to non-significant enrichment values are semi-transparent). Analysis was performed separately for genes up- and down-regulated.

C, D Genomic views of relevant TF binding to Cebpa and Gata3 loci (supporting direct regulatory interactions identified in Figs 3A,B,C). The red arrow indicates a putative enhancer element, which is bound by Ebf1 and flanked by AcK27-rich regions.
Interactions between lineage-associated transcription factors 
govern haematopoietic progenitor states (2020)
Figure 6

Figure 6 - TF network uncovers novel functions of Ebf1 and Gata3 in maintaining a multipotent, self-renewing state.

A, B Bulk RNA-Seq expression levels for Gata3 and Ebf1 in a set of isolated primary cells (data from Immgen (Heng et al, 2008)) compared to Hoxb8-FL cells. Horizontal line indicates mean expression.

C Correlation in gene expression changes following Ebf1 and Gata3 loss in Hoxb8-FL cells. Each quadrant corresponds to a set of target genes with a common regulation by Gata3/Ebf1 - I - inhibition/inhibition, II - activation/inhibition, III - activation/activation, IV - inhibition/activation. For each group the sums of scaled expression values in mouse LK/LSK or human BMMC landscapes is plotted to highlight cell types with the highest overall expression. For landscape annotation see Fig 7B andE.. Changes in expression for example genes are provided in Appendix Fig 2G-J. Blue line indicates the linear fit with shaded areas as confidence intervals.

D Schematic representation of the experiment performed by (Li et al, 2018). Ebf1 was re-expressed in Ebf1-/- pre-pro B cells thus resuming their differentiation.

E Correlation in expression changes after 24h following Ebf1 loss in Hoxb8-FL cells and re-expression of Ebf1 in Ebf1-/- pre-pro B cells (Li et al, 2018). Blue line indicates the linear fit with shaded areas as confidence intervals.

F, G Expression of the key marker genes (F) and Ebf1, Pax5, Gata3, Cebpa factors (G) along pseudotime corresponding to the B-cell differentiation trajectory in human foetal liver cells, data from (Popescu et al, 2019).
LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling (2019)
Figure 1

Figure 1. HAND2-AS1 is highly expressed in liver CSCs and its knockout promotes chemically induced HCC development.

(A) Geometric mean-centered, hierarchical cluster heat map from microarray data. 1077 annotated lncRNAs (p<0.05) were represented in liver CSC (CD13+CD133+) compared with non-CSC (CD13-CD133-) cells sorted from three HCC primary cells of three patients. Top ten upregulated lncRNAs in CSCs are shown.

(B) In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm.

(C) Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12).

(D) Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm.

(E) Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD.

(F) Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.

Data information: ** P < 0.01 and *** P < 0.001 by two-tailed Student's t-test.
LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling (2019)
Figure 2

Figure 2. HAND2-AS1 is required for the self-renewal maintenance of liver CSCs.

(A) HAND2-AS1 knockout causes a declined oncosphere-forming capacity in HCC cells. The right panel represents statistical results as means ± SD (n=3 per group). Overexpression of HAND2-AS1 (oelnc) rescued the sphere formation reduced by HAND2-AS1 deletion. Scale bar, 100 μm.

(B) CD13+CD133+ (CSC) subpopulations were detected in HAND2-AS1 knockout cells by FACS analysis. Results are shown as means ± SD (n=4 per group).

(C) Limiting diluted HAND2-AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB/c nude mice. n=12 for each group.

(D) Estimated frequency of TICs in HAND2-AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis. Data are shown as the mean and 95% confidence interval. (n=12 grafted tumors per dilution).

(E, F) Estimated frequency of TICs in HAND2-AS1 deficiency and control HCC cells during serial transplantations. Grey area indicates the 95% CI.

(G) Estimated frequency of TICs in different populations after the first transplantation (n=15 grafted tumors per dilution). Data represent the mean and 95% confidence interval.

(H) Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 KO #1 or KO #2 vectors.

(I) Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD. One-way ANOVA with Dunnett's correction for multiple comparisons.

(J) HAND2-AS1 expression levels (normalized to 18S rRNA) in livers from BALB/c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg−1 ASOs on days 15, 20, 25, 30 and 35 (n = 5 per group). Three ASOs (1, 2, 3) against HAND2-AS1 were used to treat mice and showed similar depletion effects. Results are shown as means ± SD. n=6 mice per group.

(K) Mice were killed at the 40th day after HCC cell injection, and the tumors were excised and weighed. Representative tumors are shown. Scale bar, 1 cm. Statistical data are shown as means ± SD (n = 6 mice per group) (right panel).

(L) HAND2-AS1 overexpression enhances the capacity of oncosphere formation. Scale bar, 100 μm. oe: overexpression. Right panel: Data are shown as means ± SD (n = 3 per group).

(M, N) Estimated frequency of TICs in HAND2-AS1 overexpression and control HCC cells during serial transplantations.

(O) Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 overexpression vectors.

(P) Quantification for tumor numbers one month after tumor cells orthotopically implanted to B-NSG mice. (n = 5 mice per group).

Data information: * P < 0.05 and ** P < 0.01 by two-tailed Student's t test unless indicated otherwise.
LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling (2019)
Figure 3

Figure 3. HAND2-AS1 associates with the INO80 complex and INO80 deficiency inhibits liver cancer development.

(A) Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2.

(B) Two core components of the INO80 complex were confirmed by immunoblotting.

(C) Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.

(D) HAND2-AS1 was visualized by RNA FISH, followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells. Scale bar, 100 μm.

(E) Mapping analysis of INO80-binding domains of HAND2-AS1. Schematic diagram of HAND2-AS1 full-length and truncated fragments (top panel); Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2-AS1 fragments (middle panel); Different HAND2-AS1 fragments (bottom panel).

(F) EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein. (G) Binding affinity of HAND2-AS1 with INO80 was determined by five independent EMSA assays. Non-linear regression curves were generated by GraphPad Prism. Results are shown as means ± SD.

(H) Interaction regions of INO80 with HAND2-AS1 mutations at nt 83-242 of exon 2 were detected using RNA pulldown assays. LR, loop region.

(I) Representative whole-body imaging of Huh7-Luc cells transduced with control or INO80 shRNA #1 or shRNA #2 vectors.

(J) Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD (n = 5 mice per group). One-way ANOVA with Dunnett's correction for multiple comparisons.

(K) Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).

Data information: ** P < 0.01 by two-tailed Student's t-test.
LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling (2019)
Figure 4

Figure 4. HAND2-AS1 directly binds BMPR1A promoter to initiate its expression and activates BMP signaling.

(A) Heatmap results for HAND2-AS1 or INO80 deficiency in HCC cells.

(B) Based on GSEA results, significantly changed genes due to depletion of HAND2-AS1 and INO80 were attributed to the BMP signaling in HCC primary cells. NES, normalized enrichment score; FWER, familywise error rate.

(C) ChIRP-seq analysis of HAND2-AS1 genomic binding at target sites in liver CSCs, using LacZ probes as negative control. A 10-kb interval centered on the called HAND2-AS1 peak is shown.

(D) ChIRP-seq analysis of HAND2-AS1 bound regions in the genome of liver CSCs.

(E) Representative ChIRP-seq of HAND2-AS1 binding of BMPR1A.

(F) Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls.

(G) HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.

(H) ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs.

(I) EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region.

(J) BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test.

(K) BMP signaling was examined in INO80-silenced liver CSCs by Western blot.
LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling (2019)
Figure 5

Figure 5. BMPR1A promotes liver CSC self-renewal and liver cancer development. (A) BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520).

(B) Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.

(C) Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm.

(D) BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4.

(E) BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group.

(F) Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs.

(G) Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group).

(H) Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.

(I) Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.

(J) BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).

Data information: * P < 0.05, ** P < 0.01, and *** P < 0.001 by two-tailed Student's t test unless indicated otherwise.
LncRNA HAND2-AS1 promotes liver cancer stem cell self-renewal via BMP signaling (2019)
Figure 6

Figure 6. ASOs of HAND2-AS1 and siRNAs against BMPR1A have synergistic anti-tumor effects on humanized HCC models.

(A) Schematic representation of the experimental workflow. Patient-derived HCC cells or Huh7-Luc cell lines were orthotopically transplanted into livers of B-NSG mice. Then mice were treated with scramble RNAs, ASOs for HAND2-AS1 and/or combined with siRNAs for BMPR1A.

(B) Illustration of ASO/siRNA treatment timeline.

(C) Representative whole-body imaging of Huh7-Luc cells in different groups. Mice treated with scramble ASOs and/or siRNAs obtained similar results to vehicle treatment. n≥5 mice in each group.

(D) Quantification of tumor numbers treated with ASOs and/or siRNAs after Huh7-Luc tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5).

(E) Tumors were excised from livers of PDX liver cancer models. Scale bar, 0.5 cm.

(F) Representative H&E staining in liver sections from tumor-bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC#50 and HCC#51. Scale bar, 100 μm.

(G) Representative immunohistological staining with Ki67 antibody of tumor-bearing mice treated with ASO1 and siBMPR1A. Scale bar, 100 μm. Lower panel: Quantitation of Ki67-positive cells in different groups. Data are shown as means ± SD (n=5 per group).

(H) Northern blotting of HAND2-AS1 and immunobloting of BMPR1A in tumors from PDX HCC#50 after treatment.

(I-J) Kaplan-Meier survival analysis of B-NSG mice orthotopically transplanted with PDXs HCC#50 (I) and HCC#51 (J). Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone (n = 10 per group). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.

Data information: ** P < 0.01, and *** P < 0.001 by two-tailed Student's t test unless indicated otherwise.
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 1

Figure 1. The ESCRT-III subunit CHMP4B is recruited to Galectin3-positive damaged endolysosomal membranes.

A. Representative fluorescence micrographs of HeLa cells treated with 250 μM lysosomotropic drug LLOMe or equal volume of DMSO (Ctrl) for 1 h before fixation and immunostained with Hoechst (blue), anti-CHMP4B (green), anti-GAL3 (red) and anti-LAMP1 (white) are presented. Cells treated with LLOMe show increased recruitment of ESCRT-III protein to lysosomes when compared to Ctrl cells. Number of foci per cell were quantified and are indicated as mean ± SD. Data from >86 cells per condition from three independent experiments. CHMP4B, GAL3, LAMP1 foci per cell (Ctrl versus LLOMe treatment): p=0.0398, p=0.0033, p=0.2400 (Student's t‐test), respectively.

B. HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 treated as in (A) and stained with CD63 antibody are shown. Number of foci per cell were quantified and are indicated as mean ± SD. Data is quantified from >87 cells per condition from three independent experiments. CHMP4B, GAL3, CD63 foci per cell (Ctrl versus LLOMe treatment): p=0.0111, p=0.0050, p=0.3793 (Student's t‐test), respectively.

Data information: Scale bars: 10 μm and 1 μm (inset).
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 2

Figure 2. ESCRT-I, ESCRT-II, ESCRT-III, ALIX and VPS4A are recruited to endolysosomes upon LLOMe treatment.

To screen for ESCRT proteins that are involved in the endolysosomal repair process, cells were incubated with 250 μM LLOMe for 30 min and processed for immunofluorescence. TSG101, a component of the ESCRT-I complex, and EAP30, an ESCRT-II protein, are clearly recruited to the sites of damage compared to the DMSO control (Ctrl). As shown, the ESCRT-III complex together with ALIX is recruited to damaged endolysosomes. In contrast, HRS, a component of the ESCRT-0 complex, and HD-PTP show no recruitment to damaged endomembranes. Number of foci per cell was quantified from >85 cells per condition from three independent experiments and are presented as mean ± SD. Statistical significance for number of foci per cell in Ctrl versus LLOMe treatment was determined using Student's t‐test, the p-values for which are: HRS p=0.9257, GAL3 p=0.0050; TSG101 p=0.0243, GAL3 p=0.0489 ; EAP30 p=0.0006, GAL3 p=0.0307; CHMP2A p=0.0284, GAL3 p=0.0260; CHMP4B p=0.0028, GAL3 p=0.0414; VPS4A p=0.0309, GAL3 p=0.0461; ALIX p=0.0249, GAL3 p=0.0471; HD-PTP p=0.4898, GAL3 p=0.0105.

Data information: Scale bars: 5 μm and 1 μm (inset).
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 3

Figure 3. Dynamics of CHMP4B accumulation at the damaged endolysosomal membranes depends on other ESCRT components.

HeLa cells stably expressing CHMP4B-eGFP were transfected with siRNAs against: (A) HRS, (B) TSG101, (C) CHMP2A, (D) ALIX and (E) both TSG101 and ALIX. 48 h post-transfection cells were used for live-cell imaging experiments. LLOMe was added using perfusion system.

A. Depletion of HRS does not alter the dynamics of CHMP4B-eGFP recruitment as compared to siCtrl during treatment with LLOMe.

B. Downregulation of TSG101, with two independent siRNAs causes a delay in CHMP4B-eGFP recruitment as compared to siCtrl, indicating an important role of the ESCRT-I complex in recruiting the downstream components.

C. CHMP2A depletion accumulates and stabilizes CHMP4B-eGFP positive foci.

D. siRNA mediated depletion of ALIX shows no significant change on dynamics of CHMP4B recruitment.

E. Simultaneous depletion of TSG101 and ALIX caused almost no recruitment of CHMP4B upon induction of endolysosomal damage.

Data information: The quantification graphs represent average CHMP4B foci per cell quantified from three independent live-cell imaging experiments per condition. Error bars correspond to 95% confidence intervals. Data from >56 cells per condition were analyzed in each experiment. Right panels show knockdown efficiency of siRNA oligos as detected by Western blot (*, nonspecific immunoreactivity).
ESCRT-mediated lysosome repair precedes lysophagy and promotes cell survival (2018)
Figure 4

Figure 4. ESCRTs are essential for the repair after endolysosomal damage.

A. HeLa cells stably expressing CHMP4B-eGFP were treated with 250 μM LLOMe and 75 nM Lysotracker DND-99 and fixed at different time points as indicated. After 10 min of LLOMe treatment CHMP4B is recruited whereas the number of Lysotracker positive foci is reduced. After 30 min, lysosomes gain back functionality (judging by the increased number of Lysotracker foci) and appear recovered after 1 h indicating that the ESCRT complex is able to seal the damaged endolysosomal membranes. Representative confocal images for each time point are shown. Scale bars: 5 μm.

B. Quantification graph (>250 cells per condition from four independent experiments) showing CHMP4B and Lysotracker positive foci per cell at different time points. Error bars correspond to 95% confidence intervals.

C. Quantification graph showing dynamics of Lysotracker recovery in control (siCtrl) and both TSG101 and ALIX depleted cells. HeLa cells stably expressing CHMP4B-eGFP were co-transfected with siRNAs against TSG101 and ALIX. 48 h post-transfection, cells were pre-treated for 20 min with 75 nM Lysotracker Deep-Red, which was used as a read-out. While the decrease in the number of Lysotracker spots is quickly recovered in siCtrl, simultaneous depletion of ALIX and TSG101 leads to a severe impairment in lysosomal repair. Data from >40 cells per condition from four independent live-cell imaging experiments are shown. Graph is normalized to the area occupied by the cells. Error bars correspond to 95% confidence intervals.