EMBO/SourceData huggingface dataset:
Figure 5Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Fasting and fed blood glucose 14 days post-infection.Eight-week-old male Raptor flox/flox mice (Raptorflox/flox Cre+) were fed a HFD for 12 weeks and injected with AAV8-shDOCK5 ± AAV8-Cre or AAV8-GFP (at a dose of 3 × 1011 vg / 200 μL/ mouse) via the tail-vein 14 days prior to the in vivo study. Blood glucose levels and AUC during glucose tolerance tests. Blood glucose levels and AUC during insulin tolerance tests. Glucose infusion rates (GIR). Glucose disposal rate (GDR). Hepatic glucose production (HGP). Percentage of suppression of hepatic glucose production (HGP).
Figure 6Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The mRNA and protein expression of PEPCK and G6Pase in the liver.Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. Total and phosphorylated InsR, IRS-1Ser1101 and Akt in the liver.Male Raptor flox/flox mice (8 weeks) were fed a HFD for 12 weeks and injected with AAV8- GFP or AAV8-shDOCK5 or AAV8-shDOCK5 + AAV8-Cre via the tail vein. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1 in the liver.MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The expression of PEPCK and G6Pase mRNA and protein.MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. Total and phosphorylated InsR, IRS-1Ser1101 and Akt.MPHs from Raptor flox/flox mice were infected with Ad-GFP or Ad-shDOCK5 or Ad-shDOCK5 + Ad-Cre. The lysates were immunoblotted with indicated antibodies or β-actin. The levels of DOCK5 and Raptor protein, and total and phosphorylated mTOR and S6K1.
Figure 7Rac1 GEF activation in MPHs from WT and DOCK5-/- mice.SEW2871 treatment promotes Rac1 GEF activation and eliminates the influence of a DOCK5 deficiency on Raptor / S6K1 signaling in the mouse primary hepatocytes (MPHs) of DOCK5-/- mice.Total and phosphorylated mTOR and S6K1 and the level of Raptor protein expression in plasmids expressing a WT DOCK5 or mutant DOCK5 treated-Hep1-6 cells.Coimmunoprecipitation (Co-IP) analysis of DOCK5 and Raptor expression in Hepa1-6 cells. After transfected with indicated plasmids, lysates from cells were immunoprecipitated (IP) with anti-DOCK5 antibody or normal IgG, and then immunoblotted (IB) with the indicated antibody.DOCK5 binding to Raptor fragments (445 - 887 aa) during transient expression in Hepa1-6 cells.The DOCK5 and Raptor binding sites were identified using mutant DOCK5 (DHR-2V1559A) and Raptor fragments (445 - 887 aa) based on a Co-IP assay. VA, mutant DOCK5 DHR-2; His, His-DHR-2.
Figure 1B Immunofluorescence analysis of fibronectin (green) and collagen (red) fibers on de-cellularized ECM produced by human fibroblasts. Fibers orientation was quantified using ImageJ software. Percentages indicate oriented fibers accumulated in a range of ± 21° around the modal angle. Data is represented as mean ± s.d. (n=10 random fields from 2 independent determinations)..Scale bar, 50 µm. A representative image of picrosirius red staining from 10 analyzed is shown for each condition.
Figure 2D, E Quantification of proliferation of 1205Lu (D) and SKMEL5 (E) cells plated for 48 h on plastic, Coll-1 or the indicated fibroblast-derived ECMs prior a 96 h treatment with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi. Cells were counted by Hoechst-labeled nuclei staining. Data are represented as bar plots with mean ± SEM normalized to vehicle of 3 independent experiments. (D) **P=0.015, ***P=0.0009, ****P<0.0001 and (E) *P=0.0204, ***P=0.0006, ****P<0.0001. Kruskal-Wallis followed by Dunn's multiple comparisons test.F Flow cytometry analysis of cell cycle distribution of SKMEL5 cells cultured on plastic, Coll-1 or the indicated fibroblast-derived ECMs and treated with vehicle or 2 µM BRAFi combined with 0.01 µM MEKi. The percentage of cells in different phases of the cell cycle is indicated.G Immunoblotting of protein extracts from SKMEL5 cells cultivated as described above on plastic, Coll-1 or the indicated fibroblast-derived ECMs in the presence or not of BRAFi or BRAFi/MEKi for 96 h, using antibodies against P-ERK1/2, ERK2 or cell cycle markers (P-Rb, Rb, Cyclin D1, p27KIP1, Survivin, p53). HSP60, loading control.
Figure 3B Immunohistochemical analysis of DDR1 and DDR2 levels on human melanoma tissue microarrays. Representative IHC images and quantification (right bar histograms) of DDR1 and DDR2 expression in normal skin, naevus, primary melanoma (PM) and lymph node melanoma metastases (MM). Scale bar, 100 µm. Histological scoring of the samples was performed in blinded fashion. Samples were scored as low, medium or high for DDR1 or DDR2 expression (naevus, n = 12; PM, n = 30; MM, n = 20).Immunoblotting of equal amounts of protein extracts from melanoma cell lines (C) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.Immunoblotting of equal amounts of protein extracts patient-derived short-term cell cultures (D) using antibodies against DDR1, DDR2 or markers of the melanoma cell differentiation AXL, MITF or SOX10. ERK2, loading control.
Figure 4B Immunoblotting of protein extracts from SKMEL5 cells transfected with siCTRL or the combination siDDR1#2/siDDR2#2 prior being cultivated on FRC- or MAF-derived ECMs (left and right panels, respectively) and treated with vehicle, 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi, using antibodies against the indicated proteins. HSP60, loading control.D Immunoblotting of protein extracts from 1205Lu, SKMEL5 and MM099 cells plated for 48 h on FRC-derived ECM prior a 96 h treatment with vehicle or 5 µM BRAFi in the presence or not of Imatinib (7 µM for 1205Lu, 10 µM for SKMEL5 and MM099) or DDR1-IN-1 (1 µM for 1205Lu, 5 µM for SKMEL5, 3 µM for MM099), using antibodies against the indicated proteins. HSP60, loading control.
Figure 5A Representative images of 1205 cells cultivated on collagen-I (Coll-I) or FRC-derived ECM for 48 h prior treatment with vehicle or 5 µM BRAFi or 2 µM BRAFi plus 0.01 µM MEKi for 96 h. Immunofluorescence for Phospho-DDR1 (P(Y792)-DDR1) (red; left panels) and Phospho-DDR1/2 (P(Y796)-DDR1/P(Y740)-DDR2) (red; right panels), F-actin (green) and nuclei (blue) is shown. Enlarged images of P-DDR1 and P-DDR1/2 immunostaining are shown. White arrows indicate P-DDR1 and P-DDR1/2 cell membrane linear clustering. Scale bar, 25 µm (enlarged images: scale bar, 10 µm).
Figure 6E Kaplan-Meier survival curves of mice treated with vehicle, Imatinib, BRAFi or BRAFi plus Imatinib. Median time to progression was 18, 20, 36 and 48 days, respectively. Log rank (Mantel-Cox) for BRAFi vs BRAFi/Imatinib mesylate. ****P<0.0001 and hazard ratio (log rank): 0.2403 (95% CI of ratio, 0.08123 to 0.7106).
Figure 7A Sections of 1205Lu xenografts from Fig. 6B were stained with picrosirius red and imaged under transmission light (upper panels) or polarized light (middle panels) (scale bar, 500 µm) or imaged by second harmonic generation (SHG) microscopy (lower panels) (scale bar, 50 µm) to examine collagen fiber network upon the mono or combined regimens.
Figure 8A Immunoblot analysis of protein extracts from siCTRL- or siDDR1/2-transfected SKMEL5 cells plated on FRC- or MAF-derived ECMs in the presence or not of 5 µM BRAFi, 2 µM BRAFi plus 0.01 µM MEKi for 96 h using antibodies against DDR1, DDR2, P-ERK1/2, ERK2, RelB, p100/p52 and HSP60 as loading control were used.F, G Immunoblot analysis of protein extracts obtained from SKMEL5 (F) or MM099 (G) cells plated on FRC- or MAF-derived ECMs and treated with 5 µM BRAFi in combination or not with DDR1-IN-1 (5 µM for SKMEL5 and 3 µM for MM099) or 10 µM NIK inhibitor (NIKi) for 96 h. Antibodies against P-ERK1/2, ERK2, P-Rb, Rb, Survivin, Caspase 3, cleaved Caspase 3, RelB, p100/p52 and HSP60 as loading control were used.
Figure 1Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells.Immunoblots showing expression levels of MYC, TSC1, TSC2 or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (-Tet).Relative TSC1 and TSC2 mRNA expression levels determined by qRT-PCR for high MYC (-Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± st.dev., n=3 technical replicates) *p< 0.05; **p< 0.01; statistical relevance was determined by unpaired t-test (two-tailed).qRT-PCR analysis of TSC1 mRNA levels upon MYC suppression for 24h-72h (+Tet). Immunoblots for 24h and 48h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet) the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control.Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific sh-RNAs compared to scrambled control sh-RNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6 and α-tubulin for loading control.Immunoblots of indicated proteins in P493-6 cells with high MYC (-Tet, 72 hours) or low MYC (+Tet, 72 hours) levels either treated with rapamycin or solvent.
Figure 2Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods) (the horizontal line shows the median, whiskers show maximum and minimum data points and the box represents the first to the third quartiles, n=56 fields for tumor samples and n=21 fields for control germinal centers; scale bar = 100 μm).Quantification of immunoblot analysis of TSC expression in BL (cohort 2) (n=13) compared to healthy tonsils (n=3) or reactive lymph nodes (n=3) (mean ± st. dev.) shown in Fig EV2B.
Figure 3Left graph shows the multiplication rate of P493-6 (-Tet) cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± st.dev., n=3 biological replicates). Right graph shows percentage of apoptotic P493-6 (-Tet) cells expressing scrambled control shRNA or a TSC1-specific sh-RNA determined by FACS analysis of AnnexinV/7AAD stained cells (mean ± st.dev., n=3 biological replicates).Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30pM rapamycin where indicated (mean ± st.dev., n=3 biological replicates).TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control sh-RNA or TSC1-specific sh-RNA were treated with hydroxytamoxifen to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± st.dev., n=3 biological replicates).Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control sh-RNA or a TSC1-specific sh-RNA 3 days after seeding of equal number of viable cells (determined by trypan blue exclusion; mean ± st.dev., n=3 biological replicates).Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of Rapamycin to either completely inhibit mTORC1 activity (10nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± st.dev., n=3 biological replicates); (Bl2 cells were selected for stable knockdown with Puromycin, DG75 cells without selection).Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n=8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control sh-RNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific sh-RNA (sh-TSC1b).
Figure 4TSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in P493-6 (-Tet) cells expressing scrambled control sh-RNA or TSC-specific sh-RNA, treated with 20 nM rapamycin for 12 h, or tetracycline to repress MYC for 48 h where indicatedTSC1 knockdown increases respiration in an mTORC1 and MYC dependent manner. Rate of basal oxygen consumption in response to 10 μM of the chemical uncoupler DNP to determine maximal respiration or 10 μM of the ATP synthase inhibitor oligomycin where indicated (B) (mean ± st. dev., n=6 biological replicates).Ratio of mitochondrial to genomic DNA determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).expression of cytochrome C (CYCS) or ATP5G1 mRNAs (D) determined by qRT-PCR in P493-6 (-Tet) cells expressing either scrambled control sh-RNA or a TSC1-specific sh-RNA, and treated with 20 nM rapamycin for 12 h where indicated (mean ± st.dev., n=3 technical replicates).TSC1 knockdown results in elevated ROS levels in a MYC and mTORC1 dependent manner. FACS analysis of DCF-DA stained P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA to evaluate ROS production and treated with 20 nM rapamycin for 12 h and tetracycline for 48 h where indicated (mean ± st.dev., n=3 biological replicates).TSC1 knockdown results in increased phosphorylation of SAPK/JNK. Immunoblot of SAPK/JNK Thr183/Tyr185-phosphorylation in P493-6 (-Tet) cells expressing scrambled control sh-RNA or a TSC1-specific sh-RNA. α-tubulin expression serves as loading control.Antioxidant treatment rescues cells from death caused by TSC1 knockdown. Relative viable cell number counts of P493-6 (-Tet) cells expressing scrambled control shRNA or TSC1-specific sh-RNA 3 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 10 μM of the antioxidant BHA where indicated (mean ± st.dev., n=3 biological replicates).
Figure 5Accumulation of EU-labeled (Click-It, Invitrogen) TSC1-mRNA over 3 h in high MYC (-Tet) versus low MYC (+Tet) P493-6 cells (mean ± st. dev., n=3 technical replicates).Drawing of the TSC1 promoter and luciferase-reporter constructs with the predicted E-box (CACGTG, pos. -317/-322) indicated. Relative luciferase units (RLU) of co-transfection experiments using the indicated TSC1-promoter-reporters with MYC normalized to empty pcDNA3 expression vector in HeLa cells (mean ± st. dev., n=3). Immunoblot shows MYC overexpression and α-tubulin as loading control.TSC1 mRNA turnover determined by pulse and chase (Click-It, Invitrogen) over 8 h in P493-6 cells, either treated with tetracycline to repress MYC or left untreated (mean ± st.dev., n=3 technical replicates).miR-15a expression determined by qRT-PCR in P493-6 cells treated with tetracycline for 3 days (+Tet) to suppress MYC expression or in untreated (-Tet) cells (mean ± st. dev., n=3 technical replicates).Immunoblots showing expression levels of TSC1, S6K/P-S6K and α-tubulin in P493-6 and HEK293T cells overexpressing miR-15a or a control miRNA. At the right miR-15a overexpression was determined by qRT-PCR in P493-6 and HEK293T cells (mean ± st. dev., n=3 technical replicates).Immunoblot showing TSC1 expression, S6K/P-S6K and α‑tubulin in MCF-7 cells transfected with LNA modified anti-sense miR-15a oligos or control LNAs. At the right, successful miR-15a knockdown determined by qRT-PCR in MCF-7 cells is shown (mean ± st. dev., n=3 technical replicates).Relative luciferase activities (RLU) derived from reporter constructs containing TSC1-3´UTR wild type sequences or with mutated miR-15a seed sequence binding site on co-transfection with miR-15a in MCF-7 cells (mean ± st. dev., n=3 biological replicates). The drawing at the left shows the TSC1-3'UTR with the position of the miR-15a/16-1 seed sequence and used mutation indicated.Rate of oxygen consumption in P493-6 (-Tet) cells ectopically expressing miR-15a or control-miRNA vector, under basal conditions and in response to 10 μM DNP or 10 μM oligomycin where indicated (mean ± st. dev., n=8 biological replicates).
Figure 1A, Cell viability of NS, GBM, and astrocyte cell lines measured 72 h after irradiation (fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.0001.B, Cell viability of BT308NS and their pseudo-differentiated progeny (BT308 diff) measured 24 h after irradiation (fold vs. non-irradiated cells, ctrl). *: t-test, P=0.0006.C, Flow cytometry analysis of phosphorylated histone H2AX (H2AX) at the indicated time points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: one-way ANOVA, P=0.03.D, Neutral comet assay performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail's length is proportional to DSB extent.E, Western blot showing phosphorylation of ATM (pATM) and Chk2 (pChk2), and accumulation of RAD51 after IR (5 Gy). Total ATM protein is also shown. Vinculin was used as loading control.F, qPCR of RAD51 expression measured in NS, BT308 diff, and astrocytes at the indicated time-points after IR (5 Gy, fold vs. non-irradiated cells, ctrl). Data are represented as mean ± SEM.
Figure 2A, Flow cytometry analysis of PKH-26 staining in irradiated BT308NS, 24 h after the last irradiation. The percentage of PKH-26pos cells is indicated. Ctrl: non-irradiated cells. The mean fluorescence intensity (MFI) was as follow: ctrl, 58.21; 1.5 Gy × 4, 69.35; 2 Gy × 3, 68.20; 5 Gy, 60.29.B, qPCR of SOX2 expression measured in NS 6 h after IR (5 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.02.C, LDA (sphere forming) measuring the enrichment of GSCs after IR (5 Gy). Ctrl: non-irradiated cells *: 2 test, p<0.05.E, Tumor incidence in the LDA performed in p3 mice, as described in D.F, Histogram showing the in vivo GSC frequency measured by LDA in p3 tumors. GSC frequency (95% CI) was as follows: NS-ctrl, 1/258.2 (97.67-683.9); NS-IR 1/22.1 (8.05-62.6). *: 2 test, P=1.6×10-4.G, LDA (sphere forming) measuring the in vitro frequency of GSCs in cells derived from p3 tumors. *: 2 test, P=0.0006.H, LDA (sphere forming) measuring the in vitro frequency of GSCs in cells derived from intracranial tumors generated by BT463NS, and irradiated in vivo (2 Gy × 3 days) (n = 3). Ctrl: non-irradiated tumors. *: 2 test, P=2.04×10-11. Data are represented as mean ± CI or ± SEM in B.
Figure 3B, LDA (sphere forming) measuring the GSC frequency after IR (5 Gy) in METhigh and METneg subpopulations sorted from BT308NS. *: 2 test, P=0.001.C, Flow cytometry analysis of MET in irradiated BT308NS, 24 h after the last irradiation (2 Gy × 3 days or a single dose of 5 Gy). Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells. The MFI was as follows: ctrl, 18; 2 Gy × 3, 22.77; 5 Gy, 19.49.D, Flow cytometry analysis of MET in BT308NS weekly treated with IR (2 Gy) for 6 weeks. Dotted lines: threshold to define the percentage of MET-expressing cells. Ctrl: non-irradiated cells.E, Representative immunofluorescence staining of MET (red) on tumors generated by subcutaneous injection of BT308NS, irradiated (2 Gy × 3) and explanted 72 h after the last irradiation. Nuclei are counterstained with DAPI (blue). Ctrl: non-irradiatedtumors. Scale bar, 10 µm (63× magnification). F left, Quantification of the percentage of MET-expressing cells in tumor sections represented in (E) (n = 12 HPF/group). HPF: high-power field. *: t-test, P=0.001. F right, Quantification of MET mean fluorescence intensity in tumor sections represented in (E) (n = 12 HPF/group, fold vs. non-irradiated cells, ctrl). *: t-test, P=0.00016.G, representative METimmunohistochemistry staining of matched primary and recurrent tumors. Scale bar, 50 µm (40× magnification). H, Histogram representing the number of patient displaying different percentages of MET expressing cells in their primary or recurrent tumors: negative, 0-5%; low, 6-29%; moderate, 30-69%; high, 70-100% (see also Appendix Table 4). I, Dot plot representing the MET cumulative score (MET positive cell score + MET staining intensity score) in 19 primary and recurrent tumors (see Appendix Table 4). Wilcoxon, p<0.02. Data are represented as mean ± SEM or ± CI in B.
Figure 4A, Flow-cytometry analysis of Annexin V/DAPI incorporation and MET expression in BT308NS 36 h after IR (5 Gy). Ctrl: non-irradiated cells. Blue: MET-negative cells. Red: MET-expressing cells.B, Western Blot of METneg and METhigh subpopulations sorted from BT452NS, showing activation of PARP (cleaved PARP) and Caspase 3 (cleaved casp-3) after IR (5 Gy). Vinculin was used as loading control.C, Cell viability of METhigh and METneg subpopulations sorted from BT308NS, measured 96 h after IR (1-10 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.001.D, Clonogenic assay with METhigh and METneg subpopulations sorted from BT308NS and irradiated (1-10 Gy, fold vs. non-irradiated cells, ctrl). *: t-test, p<0.05.E, Neutral comet assay in METhigh and METneg subpopulations sorted from BT308NS, performed 24 h after IR (5 Gy). Ctrl: non-irradiated cells. Comet tail's length is proportional to DSB extent.F, Western Blot of METneg and METhigh subpopulations sorted from BT308NS, showing constitutive (ctrl) and/or IR-induced phosphorylation of ATM (pATM), Chk2 (pChk2) and H2AX (H2AX), and accumulation of RAD51, 24 h after IR (5 Gy). Total ATM protein is also shown. β-actin was used as loading control.G, qPCR of RAD51 expression in METhigh and METneg subpopulations sorted from BT308NS 24 h after IR (5 Gy, fold vs. non-irradiated control cells, ctrl). *: t-test, P=0.02. Data are represented as mean ± SEM.