Fig. 5: DNA damage response (DDR) mediated by BS-DRL1 and HMGB1. | Nature Communications

Fig. 5: DNA damage response (DDR) mediated by BS-DRL1 and HMGB1.

From: Long noncoding RNA BS-DRL1 modulates the DNA damage response and genome stability by interacting with HMGB1 in neurons

Fig. 5

a Enhanced DDR of BS-DRL1 KD neurons demonstrated by western blot analysis. Primary cortical neurons were transfected with BS-DRL1 shRNA or Scr-shRNA virus, treated with vehicle or ETO for 1 h and followed by western blot with antibodies against ATM, DNA-PKcs, p-ATM (S1981), p-DNA-PKcs (S2056), and γH2AX. Tublin was used as an internal control. b–d Enhanced DDR of BS-DRL1 KD neurons demonstrated by immunofluorescence staining. Primary cortical neurons infected with indicated shRNA virus, treated with vehicle or ETO for 1 h and stained with antibodies against p-ATM and p-DNA-PKcs. Scale bar: 10 μm. The fluorescence intensity of p-ATM and p-DNA-PKcs signal in the GFP positive cells was measured and quantified (the shRNA has GFP tag). Data are presented as mean ± SD. n = 40 neurons. *p < 0.05, ****p < 0.0001, ns not significant, AU arbitrary units. e Impaired DDR of HMGB1 KD neurons demonstrated by western blot analysis with antibodies against ATM, DNA-PKcs, p-ATM, p-DNA-PKcs, HMGB1, and γH2AX. Primary cortical neurons transfected with HMGB1 shRNA or Scr-shRNA virus were treated with vehicle or ETO for 1 h and harvested for western blot. f–i Impaired DDR of HMGB1 KD neurons demonstrated by immunofluorescence staining. Primary cortical neurons infected with indicated shRNA virus, treated with vehicle or ETO for 1 h and immunolabeled with indicated antibodies (γH2AX, p-ATM, and p-DNA-PKcs). Scale bar: 10 μm. The quantification showed the fluorescence intensity of γH2AX, p-ATM, and p-DNA-PKcs. Data are presented as mean ± SD. n = 40 neurons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant, AU arbitrary units. j–k Western blot analysis of γH2AX in BS-DRL1 KD, HMGB1 KD, or BS-DRL1/HMGB1 double deficiency neurons. Primary cortical neurons from WT or BS-DRL1 KO mice were transfected with HMGB1 shRNA or Scr-shRNA virus and treated with ETO for 1 h and followed by western blot with indicated antibodies. Data are presented as mean ± SD, n = 3. *p < 0.05 ****p < 0.0001. l, m Primary cortical neurons transduced with either BS-DRL1-shRNA, flag tagged HMGB1 overexpression virus or both were treated with ETO for 1 h and processed for western blot with indicated antibodies. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, ****p < 0.0001, ns not significant. n Reduced NHEJ repair in BS-DRL1 KD neurons. Primary cortical neurons transduced with either BS-DRL1-shRNA or scr-shRNA were subjected to NHEJ reporter assay. Data are presented as mean ± SD. **p < 0.01, ns not significant. n = 3 biologically independent experiments. o Primary cortical neurons transduced with either BS-DRL1-shRNA, flag or flag tagged HMGB1 overexpression virus and DSB repair efficiency was evaluated as in (p). Data are presented as mean ± SD. **p < 0.01, ****p < 0.0001, ns not significant. n = 3 biologically independent experiments. p The assembly of HMGB1 and other DDR proteins on chromatin was examined by chromatin fractionation and western blot analysis. Primary cortical neurons transduced with BS-DRL1 shRNA or Scr-shRNA virus were treated with ETO for 1 h and processed for subcellular fractionation and western blot analysis. Data are presented as mean ± SD. ***p < 0.001, ****p < 0.0001. n = 3 biologically independent experiments. q The occupancy of γH2AX, p-DNA-PKcs, and HMGB1 at DSBs sites was assessed using CHIP assays in primary neurons transduced with BS-DRL1 shRNA or Scr-shRNA following I-Ppo-I introduction. Data are presented as mean ± SD. *p < 0.05, ***p < 0.001, ****p < 0.0001. n = 3 biologically independent samples.

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