Journal: bioRxiv

Article Title: BMI1-mediated heterochromatinization represses G-quadruplex DNA formation to maintain genomic stability during replication

doi: 10.1101/2024.12.23.630044

Figure Lengend Snippet: A. Immunofluorescence analysis of HDFs at 24hr post-transfection with shScramble or shBMI1 plasmids. HDFs were exposed or not exposed to low concentration of the DNA replication stress agents aphidicolin or hydroxyurea for 8hr before fixation. G4 structures were labeled with the 1H6 antibody (white arrows). Scale bar: 10 µm. Bottom panel: Quantification of the experiments. 3 slides/condition and 200 cells/slide. Statistical differences were analyzed using an unpaired T-test with two tails. P ≤0.05*, P ≤0.001***. All values are means ± SEM. B. Time course analysis of HDFs knockdown for BMI1 and visualized by immunofluorescence. Note that G4 structures (white arrows) are induced before 53BP1. A large DNA damage foci labeled with 53BP1 is showed in the inset (white arrow). Scale bar: 10 µm. C. Histogram showing the size distribution of 53BP1 DNA damage foci 24hr after transfection of shScramble or shBMI1 plasmids in HDFs. DDR foci were considered as large when bigger that 1.5 scare mm. Cutoff was used to define the minimal size of a DDR foci. D. Histogram showing the size distribution of 53BP1 DNA damage foci 24hr after transfection of shScramble or shBMI1 plasmids in HDFs and treated or not treated for 8hr with DMSO, DRB and/or aphidicolin. E. Histograms showing the frequency of anaphase bridge, micronuclei and mitotic catastrophe 72 hours after transfection of shScramble or shBMI1 plasmids in HDFs. Those were compared to HDFs exposed to pyridostatin for 72 hours. 3 slides/condition and 200 cells/slide. Statistical differences were analyzed using an unpaired T-test with two tails. P ≤0.05*, P ≤0.001***. All values are means ± SEM.

Article Snippet: Primary antibodies used in this study are rabbit anti-H3K9me3 (1:500, Abcam, ab8898), rabbit anti-H3K9ac (1:500, Cell Signaling, 9671S), rabbit anti-WRN (1:100, Santa Cruz, sc-5629), p-ATR, rabbit anti-53BP1 (1:100, Novus, NB100-304), mouse ψH2AX (1:1000, Millipore 05-636), rabbit anti-H2Aub (1:200, Cell Signaling, 8240S), rabbit anti-Ki67 (1:1000, Abcam, ab15580), mouse anti-PCNA (1:250, Invitrogen, MA5-11358), mouse anti-RNAPol2 (1:500, Santa Cruz, sc-47701), and mouse 1H6 antibody recognizing G4s.

Techniques: Immunofluorescence, Transfection, Concentration Assay, Labeling, Knockdown

Journal: bioRxiv

Article Title: BMI1-mediated heterochromatinization represses G-quadruplex DNA formation to maintain genomic stability during replication

doi: 10.1101/2024.12.23.630044

Figure Lengend Snippet: A. Experimental scheme, where EdU was added at 8hr (for 8 hours) or 15h30hr (for 30min). B. Immunofluorescence analysis of shScr and shBMI1 HDFs exposed to EdU. This showed that the percentage of EdU+ cells with large DDR 53BP1 foci is reduced when cells were labeled at 15h30hr and when compared to 8hr. Arrows indicate large 53BP1 foci in EdU+ and EdU-cells. N = 200 cells/group. Statistical differences were analyzed using an unpaired T-test with two tails. P ≤0.001***. All values are means ± SEM. Scale bar: 5 µm C. Immunofluorescence analysis of shScr, shBMI1, or shScr HDFs exposed to pyridostatin for 24 hours or irradiated at 10Gy. Co-localization studies revealed that only shBMI1 HDFs present a correlation between RNA Pol2 and 53BP1 foci (R = 0.15). N = 200 cells/group. Statistical differences were analyzed using an unpaired T-test with two tails. P ≤0.001***. Scale bar: 5 µm D. Quantification of experiment in (C) revealing the high percentage of 53BP1 foci that are also positive for RNA Pol2 in shBMI1 HDFs, and when compared to irradiated shScr HDFs. N = 200 cells/group. Statistical differences were analyzed using an unpaired T-test with two tails. P ≤0.001***.

Article Snippet: Primary antibodies used in this study are rabbit anti-H3K9me3 (1:500, Abcam, ab8898), rabbit anti-H3K9ac (1:500, Cell Signaling, 9671S), rabbit anti-WRN (1:100, Santa Cruz, sc-5629), p-ATR, rabbit anti-53BP1 (1:100, Novus, NB100-304), mouse ψH2AX (1:1000, Millipore 05-636), rabbit anti-H2Aub (1:200, Cell Signaling, 8240S), rabbit anti-Ki67 (1:1000, Abcam, ab15580), mouse anti-PCNA (1:250, Invitrogen, MA5-11358), mouse anti-RNAPol2 (1:500, Santa Cruz, sc-47701), and mouse 1H6 antibody recognizing G4s.

Techniques: Immunofluorescence, Labeling, Irradiation

Journal: bioRxiv

Article Title: BMI1-mediated heterochromatinization represses G-quadruplex DNA formation to maintain genomic stability during replication

doi: 10.1101/2024.12.23.630044

Figure Lengend Snippet: A-C. Immunofluorescence analysis of HDFs at 24hr post-transfection with shScramble or shBMI1 plasmids. Scale bar: 5 µm. A. Labeling with 1H6 and 53BP1 antibodies showing co-localization (arrows) in shBMI1, with R = 0.58. B. Labeling with ψH2AX and 53BP1 antibodies showing co-localization (arrows) in shBMI1, with R = 0.95. C. Labeling with PCNA and 53BP1 antibodies showing co-localization (arrows) in shBMI1, with R = 0.96. N = 200 cells/group. Statistical differences were analyzed using an unpaired T-test with two tails. P ≤0.001***. All values are means ± SEM.

Article Snippet: Primary antibodies used in this study are rabbit anti-H3K9me3 (1:500, Abcam, ab8898), rabbit anti-H3K9ac (1:500, Cell Signaling, 9671S), rabbit anti-WRN (1:100, Santa Cruz, sc-5629), p-ATR, rabbit anti-53BP1 (1:100, Novus, NB100-304), mouse ψH2AX (1:1000, Millipore 05-636), rabbit anti-H2Aub (1:200, Cell Signaling, 8240S), rabbit anti-Ki67 (1:1000, Abcam, ab15580), mouse anti-PCNA (1:250, Invitrogen, MA5-11358), mouse anti-RNAPol2 (1:500, Santa Cruz, sc-47701), and mouse 1H6 antibody recognizing G4s.

Techniques: Immunofluorescence, Transfection, Labeling

Journal: Nature Communications

Article Title: Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

doi: 10.1038/s41467-024-53877-z

Figure Lengend Snippet: A Cell cycle profile of U2OS cells using the adapted FUCCI system, with the scaled density of nuclear IMPDH2 high (top 10%, left panel) and IMPDH2 low (bottom 10%, right panel) expressing cells. B Quantification of IMPDH2 nuclear integrated intensity across the different cell cycle phases ( n G1 = 635, n S = 418, n G2M = 243; unpaired two-tailed Wilcoxon test). C Representative pictures of cells in G1 or G2M phases of the cell cycle (scale bar 25 μm). D Quantification of ɣH2AX nuclear integrated intensity across the different cell cycle phases ( n G1 = 751, n S = 387, n G2M = 375; unpaired two-tailed Wilcoxon test). Representative pictures ( E ) and quantification ( F ) of ɣH2AX foci (ɣH2AX orange, DAPI blue; scale bar 15 μm) in MDA-MB-231 (NT, n = 6081; sh1, n = 5534; sh2, n = 2916) and CAL-51 (NT, n = 16171; sh1, n = 7916; sh2, n = 15196) cell lines 72 h after knockdown of IMPDH2; unpaired two-tailed Wilcoxon test. Representative pictures ( G ) and quantification ( H ) of ɣH2AX foci (ɣH2AX orange, DAPI blue; scale bar 15 μm) in MDA-MB-231 cells treated with increasing amounts of MPA (72 h) or etoposide control (3 h) (MPA 0, n = 6086; MPA 2.5 μM, n = 6005, MPA 7.5 μM, n = 5018, MPA 15 μM, n = 3005; DMSO, n = 2996; ETO, n = 5230; unpaired two-tailed Wilcoxon test). Guanosine titration in MDA-MB 231 WT and IMPDH2 KO cells (WT: n0 = 10683, n6.25 = 10079, n12.5 = 10368, n25 = 10784, n50 = 9917, n100 = 9275, n200 = 8232, n400 = 7834, n800 = 7326, n1000 = 5874; KO: n0 = 2161, n6.25 = 3444, n12.5 = 4147, n25 = 7014, n50 = 8741, n100 = 7009, n200 = 7446, n400 = 6388, n800 = 6268, n1000 = 5807) with representative pictures ( I ) (DAPI blue, ɣH2AX orange, 53BP1 green; scale bar 10 μm) and quantification of ɣH2AX foci ( J ), 53BP1 foci ( K ) and nuclei counting ( L ) with the percentage relative to WT nuclei count without guanosine. All box plots indicate the median value (central line), interquartile range IQR (box boundaries) and up to 1.5*IQR beyond the box boundaries (whiskers). Source data are provided as a Source Data file.

Article Snippet: ; #89190S, 1:3000), RPA70/RPA1 Antibody (Cell Signaling Technology #2267; 1:50), Anti 53BP1 rabbit (Novus Biologicals #NB100-304; 1:5000).

Techniques: Expressing, Two Tailed Test, Knockdown, Control, Titration

Journal: Nature Communications

Article Title: Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

doi: 10.1038/s41467-024-53877-z

Figure Lengend Snippet: A Volcano plot of differentially expressed genes in KO-G vs WT condition (−1>log2FC > 1, FDR < 0.05); for differential expression analysis, the p -value was calculated by Wald’s Test and adjusted for multiple hypothesis by Benjamini-Hochberg criteria (FDR). Highlighted in magenta genes related to hallmark G2M checkpoint signature (MSigDB, M5901), in ochre to immune/inflammation pathways (KEGG hsa04672, hsa05323, hsa04940, hsa05332, hsa05330, hsa04658, hsa05310, hsa05150, hsa05320, hsa05321). B Heatmap of differentially expressed genes relative to WT from G2M checkpoint (Supplementary Data ), SASP (Supplementary Data ), and DNA repair hallmark (Supplementary Data ), extracted from msigdbr R package. Representative pictures ( C ) and quantification ( D ) of ɣH2AX foci (ɣH2AX orange, DAPI blue; scale bar 15 μm) in MDA-MB-231 WT, KO, KO-WT and KO-NLS without guanosine supplementation for 96 h (WT, n = 28604; KO, n = 2707; KO-WT, n = 13572; KO-NLS, n = 14449; unpaired two-tailed Wilcoxon test). Quantification ( E ) and representative pictures (53BP1 green, DAPI blue; scale bar 15 μm) ( F ) of 53BP1 foci in WT, KO, KO-WT and KO-NLS without guanosine supplementation for 96 h (WT, n = 7642; KO, n = 1983; KO-WT, n = 4707; KO-NLS, n = 6548; unpaired two-tailed Wilcoxon test). G Heatmap of differentially expressed genes relative to WT from Apoptotic (GO:0006915, Supplementary Data ), Necrosis (msigdbr, Supplementary Data ), and Necroptotic Process (GO:0070266, Supplementary Data ), GO terms from GO.db R package. H Cell death measurements in WT, KO, KO-WT, and KO-NLS treated with DMSO or etoposide 10 μM for 48 h ( n = 3 biological replicates, paired two-tailed t -test); mean values +/− SD. I Heatmap of differentially expressed genes relative to WT from the NAD + ADP-ribosyltransferase activity (GO:0003950) expanded to include SIRT1-7 family (Supplementary Data ). J Quantification of nuclear NAD + as YFP/mCherry ratio for WT, KO with guanosine 50 μM supplementation, KO-WT and KO-NLS in S-phase enriched cells (WT, n = 1149; KO, n = 1069; KO-WT, n = 578; KO-NLS, n = 645, unpaired two-tailed Wilcoxon test); outliers removed to improve visualization. All box plots indicate the median value (central line), interquartile range IQR (box boundaries), and up to 1.5*IQR beyond the box boundaries (whiskers). Source data are provided as a Source Data file.

Article Snippet: ; #89190S, 1:3000), RPA70/RPA1 Antibody (Cell Signaling Technology #2267; 1:50), Anti 53BP1 rabbit (Novus Biologicals #NB100-304; 1:5000).

Techniques: Expressing, Two Tailed Test, Activity Assay