EMBO/soda-nel huggingface dataset:
Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2 (2019)
Figure 4

Figure 4. VEGFR2 pY1212 regulates Myc and rescue of Vegfr2Y1212F /Y1212F proliferation defect by Myc overexpression

A. Immunoblot for Myc on lung lysates from wiltype (WT) and Vegfr2Y1212F /Y1212F FVB mice tail-vein injected with PBS or VEGFA followed by circulation for time points indicated. Each lane represents one individual mouse.

B. Quantification of Myc/Actin expressed as Fold change to PBS. Min, minutes of stimulation. Error bars: SD; 2-way ANOVA p=0.0058; unpaired T test, *p < 0.05, ** p <0.01. n=3 experiments.

C-D. Myc protein expression. C. Myc and Actin immunoblotting on lysates of lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. D. Quantification of Myc/Actin expressed as Arbitrary unit. Each lane (C) and each dot (D) represent one individual mouse. 2-way ANOVA p <0.0001; unpaired T test, ** p <0.01. n=5-7.

E. Baseline transcriptional level of Myc from lung isolated ECs from untreated wildtype (WT) and mutant (VEGFR2 Y1212F) C57Bl/6 and FVB mice. Each dot represents one individual mouse. 2-way ANOVA p=0.0041; Sidak's multiple comparison test, * p <0.05. n=4-5.

F-I. Proliferation of C57Bl/6 and FVB iECs. F, G. CD31 (white), ERG (red), EdU (green) and double-positive cells (yellow) in C57Bl/6 (F) and FVB (G) iECs. EdU signal was masked with ERG signal. H, I. EdU incorporation in ERG-positive ECs normalized to total ERG-positive ECs. Each dot represents 2 fields/well, one well/mouse. Scale bar, 100 µm. Error bars: SD; 2-way ANOVA p =0.0843 (H), p=0.0474 (I); unpaired T test, *p < 0.05; **p < 0.01. C57Bl/6 n=3-6 mice, FVB n=3-5mice.

J-K. Rescue of angiogenic sprouting by Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. J. Representative images of E11.5 explants from C57Bl/6 embryos taken from wildtype (WT) and Vegfr2Y1212F /Y1212F littermates with (MycOE/Cre +) and without (MycOE/Cre-) endothelial-specific overexpression of Myc. Endothelial cell marker (CD31) immunostaining highlights the vasculature in white. Embryonic tissue blacked out to highlight sprouting area. K. Quantification as fold-increase of vessel sprouting area compared to WT MycOE/Cre -. Scale bar, 250 µm. Error bars: SD; unpaired T test, ** p <0.01, **** p <0.0001. n=4-11 explants, one explant/embryo.

L-M. Rescue of Ec proliferation. L. Myc overexpression in E11.5 C57Bl/6 Vegfr2Y1212F /Y1212F explant tissue. ERG (red), EdU (green) and double-positive cells (yellow; arrows). M. Quantification of EdU/ERG double positive ECs normalized to total ECs. Scale bar, 50 µm. Error bars: SD; unpaired T test, *** p <0.001, **** p <0.0001. n=3-7 explants, one explant/embryo.
Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2 (2019)
Figure 5

Figure 5. Myc gene transcription and transcriptional activity regulation by VEGFR2 Y1212 signaling

A. GSEAs showing upregulation of genes belonging to the K-Ras, Angiogenesis and mTORC1 gene sets in (WT) compared to VEGFR2 Y1212F isolated ECs. K-Ras and Angiogenesis gene sets derived from C57Bl/6 iECs kept in basal condition; mTORC1 gene set derived from C57Bl/6 iECs treated with VEGFA for 3h. See Supplementary Table 2. Enrichment score (ES), normalized ES (NES) are shown. P-values <0.05 were regarded as significant.

B. Heat maps displaying gene regulation in gene sets shown in A. The color in the heatmap shows the gene expression value, expressed as a z-score across animals. Red color represents relative higher expression and blue color represents relative lower expression value. The z-score is calculated by (x-x')/s where x is individual gene expression, x' is the mean of the gene expression across samples and s is standard deviation of the gene expression across samples.

C-F. Myc-regulated gene expression downstream of VEGFR2 pY1212. Quantitative PCR analysis of a set of known Myc-regulated transcripts in C57Bl/6 (C, D) and FVB (E, F) adult lungs at basal (C, E) and at 1 hour after tail-vein administration of VEGFA (D, F). FASN, fatty acid synthase; Ldha and -b, Lactate dehydrogenase A and B; Acod1, Aconitate Decarboxylase 1; Ppat, Amidophosphoribosyltransferase; Pde4d, cAMP-specific 3',5'-cyclic phosphodiesterase 4D; Prkaca, cAMP-dependent protein kinase catalytic subunit alpha. Error bars: SD; 2-way ANOVA between WT and Vegfr2Y1212F /Y1212F p=0.0453 (C), p < 0.0001 (D), p=0.0018 (E), p=0.09 (F); unpaired T test, *p < 0.05, **p < 0.01, ***p < 0.001. C57Bl/6 n=8-11 mice, FVB n=9-14 mice.

Expanded View legends
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 1

Figure 1. DIS3 loss triggers genomic instability.

A. Enrichr screenshot of biological processes in which are involved the top 1% of genes whose expression varies together with the deregulation of DIS3.

B. DIS3 expression in total cell lysates of control (siCTRL) and DIS3 U2OS silenced cells (siDIS3) was analyzed by Western blot upon rescue with empty vector (EV) or DIS3 expressing vector (DIS3). Lamin B was used as the loading control.

C. Representative images of comet assays performed under alkaline conditions in U2OS cells transfected with siCTRL or siDIS3 and then infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Quantitative analysis of comet tail lengths for each condition. Plots represent three biological replicates, with more than 150 cells for each point. The analysis is performed using Casplab software, error bars indicate s.e.m. (n=3). **** p-value < 0.0001. ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.

D. Immunostaining against RPA in U2OS cells transfected with siCTRL or with siDIS3 and 48 hours later infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). The averages of foci number per cell were calculated from three independent experiments using Cell Profiler software. At least 150 cells were counted for each point, error bars indicate s.e.m. (n=3). * p-value < 0.05. ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 2

Figure 2. DIS3 silencing affects genome integrity by increasing DNA:RNA hybrids.

A. Immunostaining with S9.6 (green) and DAPI (blue) in U2OS cells, transfected for 48 hours with siCTRL or siDIS3 and then mock-treated (UNTR) or treated with RNase H or RNase III for 1 hour before fixation. Histograms show S9.6 signal intensity per cell, error bars indicate s.e.m. (n=3, biological replicates). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates).). **** = p < 0.0001, *** = p < 0.001, ns = not statistically significant, two-way ANOVA, scale bar is 10 µm.

B. Immunostaining with J2 (green) and DAPI (blue) in U2OS cells, as in panel A). More than 150 cells were counted, and error bars indicate s.e.m. (n=3 biological replicates). For all conditions, differences between siCTRL and siDIS3 were not statistically significant, two-way ANOVA, scale bar is 10 µm.

C. Dot blot analysis of DNA:RNA hybrids formation: serial dilutions (750 ng, 375 ng, 187,5 ng, 93,75 ng, 46,87 ng) of genomic DNA extracted from WT or DIS3 silenced U2OS cells and pre-treated with RNase H, RNase III or mock treated, were arrayed on a membrane and probed using the S9.6 antibody. The histogram shows the S9.6 signal quantification in 3 biological replicates, error bars indicate s.e.m., normalized on SYBR®-Gold signal (Adj. Vol.), using Image lab software. *** = p < 0.001, ** = p < 0.01, ns= not statistically significant, two-way ANOVA.

D. DRIP-qPCR analysis at four different R-loops sites, RPL13A, ACTB, ENSA, and TRIM33 in U2OS cells transduced with shScr or shDIS3 and treated or not with RNase H. Results are shown as mean + s.e.m of three independent experiments.**** = p < 0.0001, *** = p < 0.001, * = p < 0.05, two-way ANOVA.

E. Representative pictures of comet assays performed under alkaline conditions in U2OS cells transfected for 48 hours with siCTRL or siDIS3, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001, ns= not statistically significant, two-way ANOVA, scale bar is 200 µm.
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 3

Figure 3. DIS3 is required for efficient homologous recombination repair.

A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for RAD51. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). * = p < 0.05, two-way ANOVA, ns= not statistically significant, scale bar is 10 µm.

B. Design of qPCR primers for measurement of RAD51 and BRCA1 recruitment at two AsiSI sites (red arrows) located on Chromosome 1 and Chromosome 6.

C. ChIP against RAD51 was performed in DIvA cells, infected with scramble shRNA (shScr) or shRNA specific for DIS3 (shDIS3), before (- 4OHT), and after (+ 4OHT) 4OHT administration. Immunoprecipitated was analyzed by qPCR with primers specific for two AsiSI sites, on chromosomes 1 and 6. Results are expressed as the percentage of input. Error bars represent the s.e.m. of 3 independent experiments. ** = p < 0.01, *** = p < 0.001, ns= not statistically significant, one-way ANOVA.

D. Representative pictures of alkaline comet assays performed in U2OS silenced (siDIS3) or not (siCTRL) for DIS3, at sequential time points after exposure to 3 Gy irradiation. Relative comet tail moments were plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with more than 150 cells for each siRNA. **** = p < 0.0001, ** = p < 0.001, ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.

E. Growth curves of DIS3 WT and DIS3 silenced cells U2OS irradiated with 3 Gy, 72 hours after lentiviral infection with specific shRNAs. The relative growth was assessed by trypan blue exclusion at different time points after irradiation. Values were normalized against cells not irradiated. Results are shown as mean + s.e.m of three independent experiments. *** = p < 0.001, * = p < 0.05, ns= not statistically significant, two-way ANOVA.

F. Representative images of HPRT negative mutants in HT1080 cells infected with scramble shRNA (shScr) or shRNA against DIS3 (shDIS3). Histograms show the mutation frequency and the deletion length of DIS3 WT (shScr) and DIS3 depleted cells (shDIS3) after I-SceI induced DSB. Error bars represent s.e.m. (n=3). *** = p < 0.001, ns= not statistically significant Student t-test.
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 4

Figure 4. DIS3 loss induces BRCAness through DNA:RNA hybrids accumulation.

A. U2OS cells were transfected with siCTRL or with siDIS3 and 48 hours later were 3 Gy irradiated (IR) or left untreated (NO IR). Cells were fixed 4 hours after IR and stained for BRCA1. Histograms show foci number per cell, analyzed with Cell Profiler. The average number of foci per cell was calculated from three independent experiments. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3 biological replicates). ** = p < 0.01, ns= not statistically significant, two-way ANOVA, scale bar is 10 µm.

B. ChIP analysis was performed in DIvA cells, infected with scramble shRNA or shRNA against DIS3, before (- 4OHT) and after (+ 4OHT) 4OHT treatment, using an anti-BRCA1 antibody. Immunoprecipitated was analyzed by qPCR with primers specific for two AsiSI sites, on chromosomes 1 and 6. Results are expressed as the percentage of input. Error bars represent the s.e.m. of 3 independent experiments. * = p < 0.05, ns= not statistically significant, one-way ANOVA.

C. Representative images of BRCA1 staining on U2OS cells transfected with siCTRL or with siDIS3. 48 hours after transfection cells were irradiated and 4 hours later were treated with RNase H or left untreated (UNTR). Histograms show the average foci number per cell from three independent experiments analyzed with Cell Profiler. At least 200 cells were counted for each point, error bars indicate s.e.m. (n=3). ** = p < 0.01, ns= not statistically significant, one-way ANOVA, scale bar is 10 µm.

D. Representative pictures of comet assays performed under alkaline conditions in U2OS cells, silenced (siDIS3) or not (siCTRL) for DIS3 untreated (UNTR) or treated with RNase H (RNase H). Relative comet tail moments are plotted for siCTRL (black circles) and siDIS3 (red circles). Analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each siRNA. **** = p < 0.0001, one-way ANOVA, scale bar is 20 µm.

E. U2OS cells silenced (shDIS3) or not for DIS3 (shScr) were treated with Olaparib for 72 hours and viable cell counts were determined by Trypan blue exclusion. Data showed the percentage relative to the untreated cells of three independent experiments. *** = p < 0.001, ns = not statistically significant, two-way ANOVA.
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 5

Figure 5. DIS3 ensures DSBs repair by controlling DNA:RNA hybrid levels at damage sites.

A. Representative confocal images upon irradiation with the 405nm laser. U2OS cells were presensitized with BrdU for 24 hours, micro-irradiated, fixed after 10 minutes, and stained with antibodies against γH2AX and DIS3 scale bar is 10 µm.

B. Magnification image of the white box in panel A.

C. Intensity profiles of γH2AX (green) and DIS3 Sordet et al.() signals through the yellow line in panel B. Quantification is performed with ImageJ software scale bar is 10 µm.

D. ChIP using anti-flag antibody performed in DIvA cells, infected with a vector expressing DIS3 (DIS3) or an empty vector (EV), exposed (+) or not (-) to 4OHT for 4 hours. Immunoprecipitated was analyzed by qPCR with primers specific for two AsiSI sites, on chromosomes 1 and 6. Results are expressed as the percentage of input. Error bars represent the s.e.m. of 3 independent experiments. ** = p < 0.01, * = p < 0.05, two-way ANOVA.

E. ChIP was performed on DIS3 overexpressing DIvA cells treated or not with RNase H, 3 hours after exposure to 4OHT. Results are expressed as the percentage of input. Error bars represent the s.e.m. of 3 independent experiments. ** = p < 0.01, * = p < 0.05, two-way ANOVA.

F. DRIP-qPCR analysis at the AsiSI sites on chromosomes 1 and 6 in DIvA cells depleted (shDIS3) or not (shScr) for DIS3, before (-4OHT) and after (+4OHT) damage induction. Results are expressed as the percentage of input. Error bars represent the s.e.m. of 3 independent experiments. ** = p < 0.01, * = p < 0.05, two-way ANOVA.
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 6

Figure 6. DIS3 mutants show increased genomic instability, DNA:RNA hybrids accumulation, and interferon activation.

A. Representative pictures of comet assays performed under alkaline conditions in DIS3 WT MM cell lines MM.1S and RPMI-8226 (RPMI), and in DIS3 mutated MM cell lines PCM6 and OPM2, untreated (UNTR) or treated with RNase H (RNase H). Tail moments analysis was performed using Casplab software, error bars indicate s.e.m. Plots represent three biological replicates, with 150 cells for each cell line. **** = p < 0.0001 ns= not statistically significant, two-way ANOVA, scale bar is 20 µm.

B. Immunostaining with S9.6 (green) and DAPI (blue) in MM.1S, RPMI-8226, PCM6, and OPM2 cells, treated (RNase H) or not (UNTR) with RNase H for 1 hour. Histograms show S9.6 signal intensity per cell. More than 150 cells were counted, error bars indicate s.e.m. (n=3 biological replicates). **** = p < 0.0001, one-way ANOVA, scale bar is 10 µm.

C. DRIP- qPCR analysis at four different R-loops sites, RPL13A, ACTB, ENSA, and TRIM33 in RPMI-8226 and OPM2 cells infected with shScr or shDIS3 and treated or not with RNase H. Results are shown as mean + s.e.m of three independent experiments. **** = p <, ** = p < 0.01, two-way ANOVA.

D. RPMI-8226 AND PCM6 cells were treated with Olaparib for 72 hours and viable cell counts were determined by Trypan blue exclusion. Data showed the percentage relative to the untreated cells of three independent experiments. ** = p < 0.01, * = p < 0.05, ns = not statistically significant, two-way ANOVA.
Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNARNA hybrid metabolism (2022)
Figure 7

Figure 7. DIS3 mutation in patients is associated with upregulation of interferon genes and a higher mutational burden.

A. Relative mRNA expression level of interferon genes IFI6, ISG15, IFITM1, IFIT3, IFI27, MCL1, cGAS, and STING in OPM2 cells infected with a vector expressing DIS3 (DIS3) or an empty vector (EV). Values are relative to housekeeping gene expression, normalized on EV, and represent SE of three biological replicates. *** = p < 0.001, ** = p < 0.01, multiple t-tests.

B-F. Differentially expressed genes (DEGs) between mutant and WT samples; p-values are from limma and false-discovery rate (FDR) is calculated using the Benjamini-Hochberg procedure; FDR < 0.05 was used as cut off. Functional annotation analysis by IPA of the DEGs between DIS3 mutant and WT samples based on FDR cut-off. (B) The top 10 canonical pathways are represented. Percentage enrichment (bottom) and -log 10 p-value (top) indicate the number of genes belonging to the respective pathways significantly deregulated; the number of genes included in each pathway (right). In red are indicated overexpressed genes, in green downregulated. Dashed red line represents the threshold for enrichment or depletion, orange line represents gene count. (C) Volcano plot of DEGs between mutant and WT samples; labeled genes are the IFN-related genes with an FDR < 0.05 (red horizontal line) and absolute log 2-fold change above or below 0.5 (red vertical lines), respectively; additional representative IFN-related genes are also labeled. (D) Differentially Enrichment Score (ES) of IFN pathways between mutant and WT using t-test and p < 0.05 was used as cut off. Stacked bar chart showing the number of patients with low (yellow), medium (orange), or high (redSordet et al.() enrichment scores in each significant IFN pathway (left). (E) Hierarchical clustering heatmap of individual samples based on differentially ES of the IFN pathways included in (D); the expression of IFN genes in each pathway is presented. The z-score was used to indicate the direction of the respective gene expression. Within each group defined by hierarchical clustering, DIS3 mutant and WT samples are contrasted (orange and blue bars respectively). (F) Violin plot of the number of non-synonymous (NS) mutation load comparison between DIS3 mutant versus DIS3 WT performed on the entire CoMMpass dataset, including 853 MM patients, 88 presenting DIS3 mutations (all samples) and on the CoMMpass dataset, stratified according to the intensity of the IFN responsiveness (clusters C1, C2, C3). Wilcoxon rank-sum test was used for comparison between DIS3 mutant vs WT. **** = p <, ** = p < 0.01, * = p < 0.05. Black color dots represent the median in each group.
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 1

Figure 1. UBQLN4 associates with newly synthesized polyubiquitinated proteins.

(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 µM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates.

(B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µM MG-132 as well as CHX (either 10 or 25 µg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A).

(C) Schematic representation of MG-132 and CHX chase experiments in (D). During the course of MG-132 (5 µM) treatment for 4 h, CHX (10 µg/mL) was added at the indicated time points before harvesting.

(D) HeLa cells were treated as in (C), and Flag-precipitates were blotted as in (B).
UBQLN4 recognizes mislocalized transmembrane domain proteins and targets these to proteasomal degradation (2016)
Figure 2

Figure 2. UBQLN4 suppresses puromycin-induced ubiquitin-positive aggregates.

(A) UBQLN4 co-localizes with cytoplasmic aggregates after puromycin treatment. HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µg/mL puromycin for 2 h and then followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-polyubiquitin FK2 (shown in red to detect ubiquitin-positive cytoplasmic aggregates, ALIS) and anti-Flag antibodies (shown in green).

(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch’s t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).

(D) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin-induced cell death. After 48 h of UBQLN4 siRNA treatment, the cells were treated with 5 µg/mL puromycin for 15.5 h and then viability was measured. The data represent mean ± SD calculated from 3 independent experiments (n=3). P‐values were calculated by Student’s t-test compared with puromycin-treated control siRNA cells (*P < 0.01).