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. 2022 Sep 19;48(5):197. doi: 10.3892/or.2022.8412

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Copyright: © Chen et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Figure 3. — HMGB1 protein activates the AR signalling pathway by directly interacting with AR protein in PCa in vitro. (A) Following transfection with exogenous HMGB1, the expression levels of AR and HMGB1 in LNCaP were detected by western blotting. *P<0.05 compared with pLVX or sh-Scramble. (B) Following transfection with sh-HMGB1, the expression levels of AR and HMGB1 in 22RV1 cells were detected by western blotting. *P<0.05 compared with pLVX or sh-Scramble. (C) The growth abilities of LNCaP-neo, LNCaP-HMGB1, 22RV1-shScramble, and 22RV1-shHMGB1 cells were detected by CCK-8 assay. *P<0.05 compared with the 0-h group. (D) The effects of HMGB1 on the transcription levels of PSA and TMPRSS2 in LNCaP-neo, LNCaP-HMGB1, 22RV1-shScramble, and 22RV1-shHMGB1 cells were examined by RT-qPCR. *P<0.05 compared with pLVX or sh-Scramble. (E) After treatment with pLVX-shAR, AR, HMGB1, PSA, and TMPRSS2 protein expression levels in LNCaP-neo, LNCaP-HMGB1 and LNCaP-HMGB1/shAR cells were detected by western blotting. *P<0.05 compared with the control group; #P<0.05 compared with the pLVX-HMGB1 group. (F) The effects of HMGB1 and AR on the transcription levels of PSA and TMPRSS2 in LNCaP-neo, LNCaP-HMGB1 and LNCaP-HMGB1/shAR cells were examined by RT-qPCR. *P<0.05 compared with the pLVX group; #P<0.05 compared with the pLVX-HMGB1 group. (G) Following transfection with exogenous HMGB1 or sh-HMGB1, the transactivating activity of AR in LNCaP-neo, LNCaP-HMGB1, 22RV1-shScramble, and 22RV1-shHMGB1 cells was determined by a reporter gene assay. *P<0.05 compared with the pLVX group. (H and I) ChIP assay followed by RT-qPCR was used to detect the effect of HMGB1 on the ability of AR to bind to the promoters of PSA and TMPRSS2 in LNCaP and 22RV1 cells. *P<0.05 compared with the pLVX group or sh-Scramble group. (J) AR-Turbo and HMGB1-Rluc, the BRET fusion constructs, were cotransfected into PC3 cells, and the BRET signal was measured after the addition of the coelenterazine substrate. Western blotting revealed the fold changes of the expression levels of the fusion proteins. *P<0.05 compared with the AR-Turbo/HMGB1-Rluc ratio=0 group. (K) A schematic of the principle of the BRET assay. HMGB1, high-mobility group protein B1; AR, androgen receptor; PCa, prostate cancer; PSA, prostate specific antigen; TMPRSS2, transmembrane protease, serine 2; RT-qPCR, reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation; BRET, bioluminescence resonance energy transfer.