Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: RAS-induced senescence is characterized by the accumulation of irreparable DNA damage, DNA:RNA hybrids and unsuccessful loading of BRCA1. A. Immunoblot analysis of the indicated proteins in BJ-hTERT/RAS-ER cells, expressing the indicated transgenes and treated or not as indicated with 4-OHT (1µM). % of SA-β-gal positivity is indicated. B. Quantification of SA-β-gal- positive cells and S-phase entering cells (BrdU assay, BrdU pulse of 3 h) in BJ-hTERT/RAS-ER Hygro cells or BJ-hTERT/RAS-ER HDAC4 cells, treated (+RAS) or not (-RAS) with 4-OHT for the indicated days (D2 = day2, D8 = day8) to induce HRAS G12V expression. Mean ± SD; n = 4. * P < 0.05, ** P < 0.01 and *** P < 0.001 (Dunn’s Multiple Comparison Test with respect to -RAS D2). Pairwise t-test was applied to indicated comparisons. C. Representative microscopic images of SA-β-gal stained BJ-hTERT/RAS-ER Hygro cells or BJ-hTERT/RAS-ER HDAC4 cells at the indicated time (days) after 4OHT treatment (Bar = 40 μm). D. Dot blot analysis using S9.6 antibody to detect R-loops and AE-2 antibody to detect dsDNA. Around 100 and 500 ng of nucleic acids extracted from the indicated cells grown for 2 days in presence or absence of 4-OHT were treated or not with 10u of RNase H and spotted on nitrocellulose film. E. Quantification of Dot blot shown in D. Mean ± SD; n = 3. F. Schematic representation of the ChIP-seq and DRIP-seq experiments performed in the indicated cells at the indicated time after 4-OHT treatment. G. Histogram representing the percentage of genome coverage of RNA PolII Ser5, γH2AX, BRCA1, R-loops and G4s enriched peaks in the indicated cells. Mean ± SD; two independent sequencing each coming from three biological replicates. * P < 0.05, ** P < 0.01 and *** P < 0.001. (Dunn’s Multiple Comparison Test with respect to -RAS D2). Pairwise t-test was applied to indicated comparisons. H. P -value and q-value of pathway enrichment analysis performed by using MSigDB (RRID:SCR_016863) on genes associated to DNA:RNA hybrids found exclusive of BJ + RAS condition.

Article Snippet: DNA was restricted with a cocktail of 30U each of five enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI, NEB, USA) for 6 h. 10 μg of restricted gDNA was digested or not with 10 U RNase H (NEB) for 2 h at 37°C, brought to 450 μl with milliQ water and incubated O/N at 4°C by adding 50 μl of 10X binding buffer (100 mM NaPO4 pH 7, 1.4 M NaCl, 0.5% Triton X-100) with 10 μg of S9.6 antibody (MABE1095, Merck, Germany) or IgG as a control (Abcam, UK).

Techniques: Western Blot, Expressing, BrdU Staining, Comparison, Staining, Dot Blot, ChIP-sequencing, Sequencing

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: The accumulation of R-loops in OIS correlated with a reduced ability of RNase H1 to form a complex with RPA. ( A ) Around 293 cells were co-transfected with plasmid expressing GFP-RPA70 and RNase H1-FLAG as indicated. Immunoprecipitation was performed by using 1 μg of anti-FLAG antibody. 1/50 total lysate was included as input. ( B ) Co-IP experiment in BJ-hTERT/RAS-ER cells expressing or not HDAC4 as indicated, treated or not with 4-OHT to induce HRAS expression as indicated. Native lysates were immunoprecipitated with 1 μg anti-RNase H1 antibody or anti-Cherry antibody as a control. ( C ) Dot blot analysis using S9.6 antibody to detect R-loops and AE-2 antibody to detect dsDNA in BJ/hTERT cells stably expressing the indicated transgenes. Around 100 and 500 ng of nucleic acids extracted from the indicated cells were treated or not with 10u of RNase H and spotted on nitrocellulose film. ( D ) Co-IP experiment in BJ-hTERT cells expressing the indicated transgenes. Native lysates were immunoprecipitated with 1 μg anti-RNase H1 antibody or anti-IgG antibody as a control. 1/50 total lysate was included as input. ( E ) Co-IP experiment in IMR90 cells expressing the indicated transgenes. Native lysates were immunoprecipitated with 1 μg anti-RNase H1 antibody or anti-IgG antibody as a control. 1/50 total lysate was included as input. ( F ) Scheme of the experimental design for the quantitative identification of RPA70 interactors in BJ-hTERT/RAS-ER cells treated or not with 4-OHT for 2 days to reach or not a pre-senescent state. ( G ) Volcano plot showing the distribution of RPA70 interactors in BJ-hTERT/RAS-ER cells not induced to express HRAS G12V . In this graph, x -axis is plotted with log2 transformation of the fold difference between RPA70 and IgG IP; y -axis shows the-log10 transformation of the P -values for the identified proteins. RPA70, RPA32, RPA14, RNase H1 are highlighted. ( H ) Volcano plot showing the distribution of RPA70 interactors in BJ-hTERT/RAS-ER cells induced to express HRAS G12V with respect to the same cells not induced. In this graph, x -axis is plotted with log2 transformation of the fold difference between BJ + RAS and BJ-RAS; y -axis shows the -log10 transformation of the P -values for the identified proteins. ( I, J ) The in vitro assay was performed by incubating Cy5.5-labeled R-loops (I), G-loops (J) or DNA:RNA duplex (labeled both on RNA at 5′) with the indicated amounts of RPA complex or RNase H1. Native gel electrophoresis was performed to separate the various species of nucleic acids, which were schematized on the side. The fluorescence of the Cy5.5 labeled RNAs was acquired with the fluorescence reader and then the gel was stained with EtBr to detect the DNA duplexes with the transilluminator. ( K ) Histogram representing the ribonuclease efficiency of RNase H1 against G-loop substrates in the presence or absence or RPA. Data are expressed as mean ± SD; n = 4. * P < 0.05, ** P < 0.01, *** P < 0.05. Pairwise t-test was applied to indicated comparisons.

Article Snippet: DNA was restricted with a cocktail of 30U each of five enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI, NEB, USA) for 6 h. 10 μg of restricted gDNA was digested or not with 10 U RNase H (NEB) for 2 h at 37°C, brought to 450 μl with milliQ water and incubated O/N at 4°C by adding 50 μl of 10X binding buffer (100 mM NaPO4 pH 7, 1.4 M NaCl, 0.5% Triton X-100) with 10 μg of S9.6 antibody (MABE1095, Merck, Germany) or IgG as a control (Abcam, UK).

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay, Control, Dot Blot, Stable Transfection, Transformation Assay, In Vitro, Labeling, Nucleic Acid Electrophoresis, Fluorescence, Staining

Journal: Nucleic Acids Research

Article Title: RPA hyperphosphorylation hinders the resolution of R-loops and G-quadruplex-associated R-loops during RAS-driven senescence

doi: 10.1093/nar/gkag331

Figure Lengend Snippet: RPA70 overexpression in RAS-expressing cells leads to reduced R-loop levels and partial escape from oncogene-induced senescence. ( A, B ) Immunoblot analysis of the indicated proteins in BJ-hTERT/RAS-ER cells, expressing H2B-GFP or GFP-RPA70 as indicated and treated or not with 4-OHT (1 µM). ( C, D ) Quantification of SA-β-gal-positive cells and S-phase entering cells (BrdU assay, BrdU pulse of 3 h) in BJ-hTERT/RAS-ER cells, expressing H2B-GFP or GFP-RPA70 as indicated and treated or not with 4-OHT (1 µM). ( E ) Representative microscopic images of SA-β-gal-stained BJ-hTERT/RAS-ER H2B-GFP or GFP-RPA70 cells at 8 days after 4-OHT treatment (bar = 50 µm). ( F ) mRNA expression levels of the indicated SASP genes in BJ-hTERT/RAS-ER H2B-GFP or GFP-RPA70 cells at 0, 2, and 8 days after 4-OHT treatment. Mean ± SD; n = 3. Pairwise t-test was applied to indicated comparisons. ( G ) Dot blot analysis using S9.6 antibody to detect R-loops and AE-2 antibody to detect dsDNA. Around 250 ng of nucleic acids extracted from BJ-hTERT/RAS-ER H2B-GFP or GFP-RPA70 cells grown for 0, 2, and 8 days in the presence or absence of 4-OHT were treated or not with 10 U of RNase H and spotted on nitrocellulose membrane. ( H ) Quantification of R-loop signals obtained in dot blot described in G. Mean ± SD; n = 3. Dunn’s Multiple Comparison Test was applied to indicated comparisons. ( I ) Schematic illustration of the procedure for purifying proteins associated with R-loops in lysates obtained from BJ-hTERT/RAS-ER H2B-GFP or GFP-RPA70 cells expressing dRNase H1, treated or not for 2 days with 4-OHT. ( J ) Immunoblot analysis of the indicated proteins co-purified with R-loops from indicated cells treated or not for 2 days with 4-OHT, as indicated. ( K ) Quantification of the indicated proteins co-purified with R-loops from indicated cells treated or not for 2 days with 4-OHT, as indicated. In C, D, F, H, and K, data are expressed as mean ± SD; n = 3. Dunn’s Multiple Comparison Test was applied to indicated comparisons. * P < 0.05, ** P < 0.01, *** P < 0.05.

Article Snippet: DNA was restricted with a cocktail of 30U each of five enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI, NEB, USA) for 6 h. 10 μg of restricted gDNA was digested or not with 10 U RNase H (NEB) for 2 h at 37°C, brought to 450 μl with milliQ water and incubated O/N at 4°C by adding 50 μl of 10X binding buffer (100 mM NaPO4 pH 7, 1.4 M NaCl, 0.5% Triton X-100) with 10 μg of S9.6 antibody (MABE1095, Merck, Germany) or IgG as a control (Abcam, UK).

Techniques: Over Expression, Expressing, Western Blot, BrdU Staining, Staining, Dot Blot, Membrane, Comparison, Purification