Fig 1: Maspin-mediated downregulation of androgen receptor (AR) was contributed to chemotherapy efficacy. (A) Prostate cancer LNCaP cells were treated with 5′-Aza at indicated concentration for 48 h. Then total cell lysates were harvested and western blotting (WB) was conducted to examine the poly (ADP-ribose) polymerase (PARP) cleavage and the level of p-H2AX, maspin, AR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The level of GAPDH was used as equal loading control. (B) Prostate cancer 22RV1 cells were treated with 5′-Aza at indicated concentration for 48 h. Then total cell lysate was harvested and WB was conducted to examine the PARP cleavage and the level of p-H2AX, maspin, AR, and GAPDH. The level of GAPDH was used as equal loading control. (C) Prostate cancer LNCaP cells were treated with enzalutamide (5 μM) and MS-275 (1 μM) for 48 h. Then total cell lysates were harvested and WB was conducted to examine the PARP cleavage and the level of p-H2AX, maspin, AR, and GAPDH. The level of GAPDH was used as equal loading control. (D) Prostate cancer 22RV1 cells were treated with enzalutamide (5 μM) and MS-275 (1 μM) for 48 h. Then total cell lysate was harvested and WB was conducted to examine the PARP cleavage and the level of p-H2AX, maspin, AR, and GAPDH. The level of GAPDH was used as equal loading control. (E) The overexpression of maspin clone 22RV1-M7, M11, and the control clone 22RV1-Neo cells were treated with MS-275 (1 μM) and AR antagonist enzalutamide (5 μM) for 48 h. Then total cell lysates were harvested and WB was conducted to examine the PARP cleavage and the level of p-H2AX, maspin, AR, and GAPDH. The level of GAPDH was used as equal loading control.
Fig 2: SERPINB5 prevents X-ray radiation–induced NPC cell apoptosis. a Flow cytometry analysis of Annexin V and PI staining in HONE1 cells with TRIM21 GOF or LOF after X-ray radiation. Annexin+PI− cells were evaluated and quantified. b, c Quantification of apoptotic HONE1 b and 5-8F c cells. d Absorbance intensity of SERPINB5 GOF (top) and LOF (bottom) tumor cells and their counterpart controls in mice (n = 5 for each group). The tumors were evaluated 2 and 3 weeks, respectively, after implantation, and the mice received radiotherapy (2 Gy daily and a total of 12 Gy) after 2 weeks. e, f Absorbance intensity analysis of the tumors in mice. g GFP expression in HONE1 cells with SERPINB5-V2A-GFP overexpression or TRIM21 knockout. h Flow cytometry analysis of GFP+ cell percentages in HONE1 and 5-8F cells. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 3: Maspin repressed the expression of AR. (A) Overexpression of maspin in prostate cancer 22RV1 cell was conducted by ectopic recombinant maspin gene transfection as described in the section of Materials and Methods. The cells transfected with empty vector was used as Neo control. Then western blot was utilized to exam the expression of maspin, AR, and internal loading control of GAPDH in clone cells (a). Meanwhile, real-time PCR was used to evaluate the relative mRNA level of maspin, AR, NKX3.1, and TMPRSS2. The data are presented as an average of three repeats (b). (B) Prostate cancer LNCaP cells were transiently transfected with maspin siRNA as described in the section of Materials and Methods. The cells transfected with scramble RNA or solution vehicle were used as controls. Then the expression of maspin, AR, and internal loading control of GAPDH in clone cells was analyzed by WB. Three of maspin downregulated clones were designated as si#1, si#2, and si#3 (a). Meanwhile, qRT-PCR was used to evaluate the relative mRNA level of maspin, AR, NKX3.1, and TMPRSS2 in si#1, si#2, and Scr control after normalization with internal GAPDH mRNA. The data are presented as an average of three repeats (b). (C) The engineered PC3-AR18 cell clone with AR overexpression was established as described in the section of Materials and Methods. Then PC3-AR18 was transiently transfected with maspin siRNA as also described in the section of Materials and Methods. The cells transfected with scramble RNA was used as a control. Then the expression of maspin, AR, and internal loading control of GAPDH in clones was analyzed by WB. Four of maspin downregulated clones were designated as si#1, si#2, si#3, and si#4 (a). Meanwhile, real-time PCR was used to evaluate the relative mRNA level of maspin, AR, NKX3.1, and TMPRSS2 in si#1, si#2, and Scr control after normalization with internal GAPDH mRNA. The data are presented as an average of three repeats (b). The bars represent the SE The p values were obtained by one-tailed matched pair Student’s t tests (*compared with Neo group, p < 0.001).
Fig 4: SERPINB5 is indispensable for TRIM21-mediated GMPS–TP53 repression after radiation. a Co-immunoprecipitation following western blotting in NPC cells with SERPINB5–HA and TRIM21–MYC overexpression. b Immunofluorescence staining analysis of SERPINB5 and TRIM21 in NPC cells. c Western blot detection of SERPINB5 expression in normal NP69 cell line and in NPC cell lines. d Western blot detection of GMPS and TP53 in NPC cells with TRIM21 GOF or SERPINB5 LOF. e Co-immunoprecipitation using anti-HA antibody in NPC cells with SERPINB5–HA and GMPS–FLAG overexpression. f Immunofluorescence staining to detect the co-localization of GMPS and SERPINB5 in NPC cells with or without ionizing radiation. g GMPS expression in immune-purified protein by anti-HA antibody from NPC cells with or without TRIM21 LOF. h GMPS expression in immune-purified protein by anti-MYC antibody from NPC cells with or without SERPINB5 LOF. i GMPS expression in NPC cells with TRIM21 or SERPINB5 overexpression. j Immunofluorescence staining of overexpressed GMPS and ubiquitin in HONE1 cells with or without ionizing radiation. The localization of GMPS and was evaluated in condition of SERPINB5 or TRIM21 LOF. For the co-immunoprecipitation assay, MG132 was added into the cell medium before cell harvesting to avoid GMPS degradation. *P < 0.05, **P < 0.01, ***P < 0.001
Fig 5: SERPINB5 expression increases in radioresistant NPC patients. a, b Immunohistochemistry staining of SERPINB5 and GMPS in radiosensitive a and radioresistant b patients. Based on the staining intensity, the images are divided into three grades from weakest to strongest (from 1 to 3, respectively)
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