Fig 2: Inhibition of IR-mediated cell death by CTSS. a MCF7 cells were transfected with a control or WT-BRCA1. Western blots of cleaved-PARP and BRCA1 expression were performed (upper). Cell death was evaluated by PI staining after 12, 24, and 48 h of 10 Gy IR (bottom). b MCF7 cells were transfected with si-CTSS or were treated with VBY-036 (10 μM). Western blots of cleaved-PARP and BRCA1 expression were performed (upper). Prevalence of cell death was evaluated by PI staining after 48 h of exposure to 10 Gy (bottom). c MDA-MB-436 cells with or without WT-BRCA1 were treated with si-CTSS or VBY-036 (10 μM). After 48 h of 10 Gy IR, Western blotting (left) or PI staining (right) was performed. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control. The graphs depict the mean ± SD of PI-positive cells. *p < 0.05 and **p < 0.01 (ANOVA)

Fig 3: CTSS interacts with the BRCT domain of BRCA1 and inhibits BRCA1 protein expression. a Constructs of V5-CTSS and V5-C25A (active site of Cys25 mutated to Ala) were transiently transfected into Flag-BRCA1 transfected MCF7 cells, and cell extracts were subjected to immunoprecipitation (IP) and immunoblotting (IB). b Schematic presentation of BRCA1 domain, including RING, Rad51 interacting, and BRCT domains (upper). MCF7 cells were transfected with BRCA1 fragments (F1 to F6) encoding Myc with or without full-length V5-CTSS and analyzed by Western blotting (bottom). c Constructs of V5-CTSS and V5-C25A were transiently transfected into Flag-BRCT transfected MCF7 cells, and cell extracts were subjected to immunoprecipitation (IP) and immunoblotting (IB). d Schematic structure of BRCA1, BRCT, and BRCT deletion construct (ΔBRCT). MCF7 cells were transfected with WT-BRCA, BRCT with and without WT-CTSS, C25A and analyzed by Western blotting. e MCF7 cells were transfected with WT-BRCA and ΔBRCT constructs with and without WT-CTSS or sh-CTSS and analyzed by Western blotting. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control

Fig 4: Hmgb2 mediates microglia pro-inflammatory response in stroke. (A) representative images show the expression of Hmgb2-SI (red, SI-tdT) and Hmgb2-I (red, I-tdT) in the cortex at 18 days after the injection of the AAV-PHP.eB-Hmgb2-SI/tdT or the AAV-PHP.eB-DIO-Hmgb2-I/tdT virus particles into the tail vein of the Cx3cr1-Cre mice. The sections are stained with anti-Iba1 (green). (B) the numbers and the soma size of the Iba1-labeted cells in the indivudal mice (circles) and their averages per group (triangles)at 18 days after the injection of AAV are plotted (ns = no significantly differences). (C) Hmgb2-I inhibits the Hmgb2 expression in microglia. The microglia lysates are prepared from mice expressing Hmgb2-SI or Hmgb2-I at 1, 2, or 3 days after operation with sham or stroke and blotted with anti-Hmgb2 or anti-α-tubulin, as indicated. (D) Relative expression (R.E) levels (defined by normalizing the band intensities of anti-Hmgb2 blots to the respective α-tubulin) in the individual mice (circles) and their averages per group (triangles) are plotted. Data are mean ± SEM (n = 5 mice per group, ns = no significantly differences, *p = 0.040; **p = 0.003; ***p < 0.0001 between Hmgb2-SI and Hmgb2-I, t-tests). (E) AAV-PHP.eB -DIO-Hmgb2-SI/tdT virus (green symbols) or AAV-PHP.eB -DIO-Hmgb2-I/tdT (blue symbols) virus particles or saline (pink symbols) were injected into the tail vein of the Cx3cr1-Cre mice. 18 days after the injection, mice were operated with sham or stroke. 24 or 72 hours after the operation, the ratios of IL-1, Cscl-16, Ctss, IL-6 and TNF-a in the extracellular space of stroke mice versus sham mice (stroke/Sham) are analyzed and plotted by the individual mice (circles) and their averages per group (triangles). Data are mean ± SEM (n = 5 mice per group, **p = 0.0041, ***p < 0.0001 between Hmgb2-SI and Hmgb2-I, t-tests). (F) the ratios of IL-10 in the extracellular space of stroke mice versus sham mice (stroke/Sham) are analyzed and plotted by the individual mice (circles) and their averages per group (triangles). Data are mean ± SEM (n = 5 mice per group, F (3,16) = 9.506, ns = no significantly differences, **p = 0.0038, 0.0011, 0.0054, BF ANOVA).

Fig 5: CTSS enhances BRCA1 downstream functions. a Promoter reporter construct gadd45 was co-transfected with BRCA1, ΔBRCT, CTSS, si-Con, si-BRCA1, or si-CTSS into MCF7 cells. Cells were collected and subjected to luciferase assay. b MCF7 (upper), MDA-MB-231 (middle) and MDA-MB-436 (bottom) cells were co-transfected with WT-CTSS or si-CTSS either the si-Con or si-BRCA1 as indicated. c CTSS and BRCA1 protein expression following treatment of MCF7 or MDA-MB-231 cells with CTSS specific inhibitor VBY-036 (10 μM) (upper). Promoter activity gadd45 was following treatment of MCF7 or MDA-MB-231 cells with CTSS-specific inhibitor VBY-036 or si-CTSS (bottom). d MCF7 cells were treated with IR (10 Gy) after transfection with WT-BRCA1, WT-CTSS, si-CTSS, or si-BRCA1 constructs and Western blotting was performed. Protein levels were quantified using Image J software and data are expressed as the fold change relative to the negative control. Normalized luciferase activities were referred to the activity of extracts. Graphs represent Mean ± SD of three experiments. *p < 0.05 and **p < 0.01 (ANOVA)