Fig 1: The influence of DNAPKcs and XLF on the sizes of deletions and insertions at EJ junctions, and on a +1 insertion EJ event likely caused by staggered Cas9 DSBs.a Shown is the frequency of deletion sizes for parental cells with or without M3814, PRKDC-KO, and XLF-KO cells for the deletion events shown in Fig. 3d. n = 3 biologically independent transfections. Statistics with unpaired two-tailed t test using Holm–Sidak correction. 1–5 nt deletion: ***P = 0.000475, **P = 0.00506, *P = 0.0447. 6–10nt deletion: Parental DMSO vs 500 nM M3814 ***P = 0.000937, Parental DMSO vs. PRKDC-KO ***P = 0.000596, *P = 0.0247. 11–15 nt deletion: **P = 0.00824. 16–20 nt deletion: ***P = 0.000189, *P = 0.0491. n.s.= not significant. b Shown is the frequency of deletion sizes for parental and XLF-KO cells with or without M3814 for the deletion events shown in Fig. 3e. n = 3 biologically independent transfections. Statistics as in (a). 1–5nt deletion: ****P < 0.0001, Parental EV DMSO vs. 500 nM M3814 ***P = 0.000473, XLF-KO XLF-WT DMSO vs. 500 nM M3814 ***P = 0.000473, **P = 0.00374. 6–10nt deletion: ****P < 0.0001, Parental EV DMSO vs. 500 nM M3814 **P = 0.00127, XLF-KO XLF-WT DMSO vs. 500 nM M3814 **P = 0.00127, *P = 0.041. 11–15nt deletion: n.s.= not significant. 16–20 nt deletion: Parental EV DMSO vs. 500 nM M3814 ***P = 0.000189, XLF-KO XLF-WT DMSO vs. 500 nM M3814 ***P = 0.000274, XLF-KO XLF-K160D DMSO vs. 500 nM M3814 ***P = 0.000121, *P = 0.0104. >20 nt deletion: †P = 0.0478 but not significant after correction (unadjusted P-value), n.s.= not significant. c Shown is the frequency of insertion sizes for parental cells with or without M3814, PRKDC-KO, and XLF-KO cells for the insertion events shown in Fig. 3d. n = 3 biologically independent transfections. Statistics as in (a). 1 nt insertion: **P = 0.00147. 2 nt insertion: **P = 0.00692. =3 nt insertion: *P = 0.0446, †P = 0.0257 but not significant after correction (unadjusted P-value). n.s.= not significant. d DNAPKcs is less important to promote EJ with 1 nt insertions (+1 EJ) compared to XLF. Shown is the EJ7+1-GFP reporter to measure +1 EJ, which is the same chromosomal reporter as in Fig. 1, but using a different 5' sgRNA. n = 6 biologically independent transfections. Statistics as in (a). ****P < 0.0001. Data are represented as mean values ± SD.
Fig 2: SSBP1 is inhibited by pterostilbene to suppress DNA-PK/p53 pathway in the alleviation of high fructose-induced glomerular podocyte ferroptosis. (A) Rats were assigned into control group with a standard water and high fructose group fed with 10% fructose solution(W/V) for 16 weeks. After 8-week fructose intake, high fructose-fed rats were randomized into 5 subgroups, receiving water (vehicle), 10, 20 and 40 mg/kg pterostilbene, and 4 mg/kg pioglitazone for the next 8 weeks. Western blot analysis of the protein levels of SSBP1, p-DNA-PKcs(S2056), p-p53(S15), nucleus p53 and SLC7A11 in rat glomeruli (n = 6 per group). (B) Podocytes were cultured with or without fructose (5 mM), pterostilbene (10 µM) and pioglitazone (10 µM). Western blot analysis of the protein levels of SSBP1, p-DNA-PKcs(S2056), p-p53(S15), nucleus p53 and SLC7A11 in podocytes (n = 6 per group). (C) Lipid peroxidation in podocytes was analyzed by C11-BODIPY (581/591) staining and measured by flow cytometer (n = 6 per group). (D–E) The MDA level was measured by assay kit in rat glomeruli and podocytes (n = 6 per group). (F) Cell death was analyzed by TUNEL staining in rat glomeruli. Green fluorescence in pictures represented positive signals (cell death) (scale bras: 20 µm) (n = 3 per group). (G) Podocyte death was analyzed by propidium iodide staining and measured by flow cytometer (n = 6 per group). Data are plotted as mean ± SEM. P-values were acquired by one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001 vs control vehicle group, +P < 0.05, ++P < 0.01, +++P < 0.001 vs fructose vehicle group.
Fig 3: Chromatin decondensation in response to DNA damage at early time points requires DNA-PKcs kinase activity. (A) IR-induced chromatin relaxation is attenuated in the HCT116 DNA-PKcs KD/- and -/- cells. The HCT116 DNA-PKcs +/-, -/-, and KD/- cell lines were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Chromatin decondensation was then determined by examining micronuclease (MNase) accessibility. Nuclei were processed and the DNA was visualized by resolving it via agarose gel electrophoresis. Panels on the right show quantified signal as the percent of total for each lane across a distance from the well to the end of gel. (B) ATM kinase activity is not required for the initial chromatin relaxation after IR-induced DNA damage. The HCT116 DNA-PKcs +/- and KD/- cell lines were mock-treated or incubated for 2 h prior to irradiation with 10 µM KU60019 to inhibit ATM and then the cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Samples were processed and quantified as described in (A). (C) Inhibition of DNA-PKcs suppresses IR-induced chromatin decondensation in ATM-deficient AT5 cells. The AT5 cells were mock-treated or incubated for 2 h prior to irradiation with 10 µM NU7441 to inhibit DNA-PKcs and then the cells were mock-treated or irradiated with a dose of 10 Gy and allowed to recover for 10 min. Samples were processed and quantified as described in (A). All data (A–C) are a representative image of three independent experiments.
Fig 4: Resistance to irradiation in vivo conferred byRAD18 in esophageal squamous cell carcinoma. (A, B) The images and growth curve showed the volume from ECA-109 cell-derived (ECA-109 NC and ECA-109 RAD18) tumor xenografts in nude mice treated with radiation therapy. The tumor volumes were expressed as the mean ± standard error of the mean (SEM) of three separate experiments. (C, D) Hematoxylin and eosin(H&E) and immunohistochemical (IHC) staining analyses of RAD18 and p-DNA-PKcs (S2056) in the indicated tumor tissues from the xenografts. Counts or scores of IHC are expressed as the mean ± SEM. ** p < 0.01, *** p < 0.00
Fig 5: CXCL1 enhanced DNA damage repair in a SOD1–ROS-axis-dependent manner. (a and b) The ROS level following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or CAF medium with or without 500 ng/ml CXCL1 antibody or 400 nM CXCR2 inhibitor SB225002 for 24 h, by immunofluorescence analysis. Magnification: × 10. (c and d) The fold change of SOD1 mRNA level before or after radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody or 400 nM CXCR2 inhibitor SB225002 for 24 h, by qRT-PCR analysis. *P<0.05, **P<0.01. (e) The expression of SOD1 protein following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 500 ng/ml CXCL1 antibody for 24 h, by western blotting analysis. GAPDH was used as a loading control. (f) The expressions of ?-H2AX, p-ATM, Rad50, p-Rad50, p-Chk2, Ku80, DNA-PKcs, p-DNA-PKcs and SOD1 following radiation in KYSE-150 and in KYSE-30 that were cultured in normal medium or in CAF medium with or without 100 ng/ml human SOD1 protein for 24 h, by western blotting analysis. GAPDH was used as a loading control
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