Fig 2: hnRNP H/F drive genomic instability and therapy resistance.a Immunofluorescence experiments in LN18 cells using the ?-H2AX, 53BP1 antibodies and DAPI staining. Mean intensities of ?-H2AX and 53BP1 in 2322 cells were plotted; the bottom and top of the box present the first and third quartile, respectively; the band inside the box shows the mean and the whiskers show the upper and lower extremes. Statistical significance was performed on the full cell populations. n = 2322 cells examined. Shown is a single representative field from one experiment over n = 2 independent experiments. For ?-H2AX: ***P-value = 4.26e-10, for 53BP1: ***P < 2.2e-16 (two-sided Mann & Whitney test). b Western blot analysis of ?-H2AX in LN18 cells treated with dose scale of carboxypyridostatin (cPDS) for 24 h. Shown is a representative result from n = 3 independent experiments. c Quantification of the ?-H2AX levels in LN18 treated with cPDS normalized to GAPDH levels and plotted relatively to the untreated condition. Data are presented as mean values ± SEM of n = 3 independent experiments, P-value = 0.0157 and P-value = 0.0457 for the 2 µM and 10 µM cPDS treatment respectively (two-sided paired t-test). d Quantification of DNA repair kinetics by western blot analysis of ?-H2AX after 4 Gy ?-irradiation in LN18 cells treated with control (siCtr) or hnRNP H/F (siH/F) siRNAs. Shown is a representative result from n = 2 independent experiments. e Plating efficiency assays measuring the cell survival fraction in LN18 treated with siCtr or siF siRNAs and submitted to a radiation dose scale. Data are presented as mean values ± SEM of 6 wells, P-value = 0.0003 and P-value = 0.0006 for the 2 Gy and 4 Gy dose, respectively (two-sided paired t-test). f Quantification of DNA repair kinetics by western blot analysis of ?-H2AX after temozolomide (TMZ) treatment in LN18 cells transfected with an empty plasmid (pICE) or a plasmid expressing Flag-hnRNP H/F. Shown is a representative result from n = 2 independent experiments. For all panels, source data are provided as a Source Data file.

Fig 3: Expression analysis of RAD51 and its paralogs in PTEN-mutated/deficient GBM. (A) Expression analysis of RAD51 and its paralogs stratified by the PTEN status from The Cancer Genome Atlas (TCGA) dataset. Box-and-whisker plots of log2-transformed gene expression levels in glioblastoma tissue specimens from TCGA sorted by the PTEN mutational status denoted as wild-type (WT; n = 95) or mutant/deletion (mut/del; n = 50). An unpaired Wilcoxon rank-sum test was conducted between PTEN WT and mut/del expression values for each gene, resulting in the displayed p values. (B) Expression analysis of RAD51 and its paralogs stratified by the PTEN status from patient-derived GBM brain tumor initiating cells (BTICs). Box-and-whisker plots of log2-transformed gene expression levels in BTICs from the SKS dataset sorted by the PTEN mutational status denoted as wild-type (WT; n = 7) or mutant/deletion (mut/del; n = 6). An unpaired Wilcoxon rank-sum test was conducted between PTEN WT and mut/del expression values for each gene, resulting in the displayed p values. (C) Western analysis of the BTICs derived from primary and recurrent GBM patients. Each BTIC line is derived from a GBM patient with an independent PTEN mutation (PTEN status) predicted to abrogate the PTEN protein stability/function based on the Gene Analysis Toolkit (GATK) sequence analysis (https://gatk.broadinstitute.org/hc/en-us; Broad Institute, Cambridge, MA, USA) and the previous studies [58]. RAD51 is expressed in all PTEN-mutant/deficient primary GBM BTICs, while the DNA damage generated by topotecan (TPT; 10 µM at 37 °C for 2 h) induces robust phosphorylation of 53BP1 (p-53BP1S1778) indicating active cellular non-homologous end joining (NHEJ) activity. The presence of phosphorylated ATM (p-ATMS1981) and KAP1 (p-KAP1S824) is indicative of induction of the DNA damage response. Irradiation of immortalized lymphoblastoid cells (LCLs) from normal (WT) and ataxia–telangiectasia (A–T) patients serve as ATM-proficient and -deficient control lines, respectively, to track ATM/KAP1-mediated DNA damage signaling.

Fig 4: NRF2 affects radiosensitivity by modulating DNA repair independently of ROS. (A) Western blotting of NRF2 in A549, A549-NRF2KO and A549-NRF2KO+Flag-NRF2 cells. (B) Colony-formation assay with A549, A549-NRF2KO and A549-NRF2KO+Flag-NRF2 cells. A549-NRF2KO cells were generated using CRISPR-Cas9 and transfected with a Flag-NRF2 expression vector to generate A549-NRF2KO+Flag-NRF2 cells. Cells were pretreated with 5 mM NAC for 1 h and then exposed to 0, 2, 4 or 6 Gy IR. Data are means ± S.E.M. (n = 3). ***P = 0.001. (C) A549 cells transfected with siCtrl or siNRF2 were pretreated with NAC for 1 h and exposed to 8 Gy Cs137 ?-rays. At 24 h post-irradiation, the percentage of apoptotic cells was determined by co-staining with FITC-Annexin V and PI. Data are means ± S.E.M. (n = 3). ***P = 0.001, ns: no significance. (D) A549 cells transfected with siNRF2 or siCtrl were pretreated with NAC for 1 h and exposed to 8 Gy Cs137 ?-rays. ?H2AX foci were counted at 1, 8, and 24 h post-irradiation. Scale bars, 25 µm. Data are means ± S.E.M., with >150 cells were counted in three independent experiments. **P = 0.01, ***P = 0.001. (E) A549 cells transfected with siCtrl or siNRF2 were pretreated with NAC for 1 h and exposed to 8 Gy Cs137 ?-rays. 53BP1 foci were counted at 1, 8, 12 and 24 h post-irradiation. Scale bars, 50 µm. Data are means ± S.E.M., with more than 150 cells were counted in three independent experiments. ***P = 0.001. (F) Western blotting of NRF2 in A549 cells treated with shScr#1, shNRF2#1, shScr#2 and shNRF2#2. (G) A549 cells treated with shScr#1, shNRF2#1, shScr#2 or shNRF2#2 were pretreated with or without 5 mM NAC and exposed to 8 Gy Cs137 ?-rays. The percentage of cells with micronuclei (MN) was determined at 24 h post-irradiation. Data are means ± S.E.M. (n = 3). *P = 0.05, **P = 0.01, ***P = 0.001.

Fig 5: SETD2 promotes 53BP1 recruitment to DNA damage sites.(A) 53BP1-GFP transfected U2OS cells were damaged by laser irradiation of the indicated nuclear region. The dynamics of 53BP1-GFP during the DNA damage response on control and SETD2-depleted cells was monitored by live cell imaging for 15 min after laser irradiation. One representative experiment from over 50 individual cells recorded is shown. (B) Control and SETD2 RNAi-depleted U2OS cells were transfected with a 53BP1-GFP expression plasmid. Western blot analysis was performed with antibodies against GFP (to detect 53BP1-GFP), H3K36me3, total histone H3, and a-tubulin. Molecular weight markers (KDa) are shown on the left. (C) SETD2-GFP transfected U2OS cells were lysed and processed for western blot with antibodies against GFP (to reveal SETD2-GFP), H3K36me3 and a-tubulin. Molecular weight markers (KDa) are shown on the left. (D) Live-cell images of SETD2-GFP dynamics were recorded upon laser-induced DNA damage of the indicated nuclear region of U2OS cells during 15 min (E) H3K36me3 was immunoprecipitated from MNase-digested U2OS cell extracts and each of the four nucleosomal histones was detected by immunoblotting of the SDS-PAGE resolved complexes. (F) Nuclear co-immunoprecipitations before and 6 hr after NCS treatment were performed on U2OS cells using the indicated antibodies (I.P.: 53BP1 and H3K36me3). The Input lane represents total cell lysates and (-) denotes the negative control immunoprecipitation (beads only). Purified complexes were resolved by SDS-PAGE and blotted with antibodies against ?H2AX or H3K36me3.DOI: http://dx.doi.org/10.7554/eLife.02482.004