Fig 2: Ectopic overexpression of ARv7 upregulates MALT1 expression to enhance NF-κB activation in PC-3 and 22Rv1 cells. An immunoblot assay of the expression of ARv7, MALT1, PSA, NDRG1, and β-actin in 22Rv1 (A) and PC-3 (B) cells. The quantitative data of the immunoblot assays were presented as the mean fold induction (±SE, n = 3) of the target proteins/β-actin relative to the mock-transfected group. The reporter activity of the MALT1 reporter vector was cotransfected with various concentrations of the ARv7 expression vectors, as indicated, for 24 h in the 22Rv1 (C) and PC-3 (E) cells. The data are presented as mean percentages (±SE, n = 6) relative to the control vehicle-treated group. The expression of ARv7, MALT1, IκBα, phospho-IκBα, p65, p50, Lamin B1, and GAPDH in the 22Rv1 (D) and PC-3 (F) cells after the separation of the nuclear and cytoplasmic fractions, as indicated, were determined by immunoblot assays. The numbers indicate the ratio of target proteins/GAPDH or Lamin B1 in relation to the mock-transfected group. The NF-κB (p65)-binding activity in the 22Rv1 (G) and PC-3 (H) cells after stably transfecting with ARv7. ** p < 0.01.

Fig 3: Androgen does not affect MALT1 expression in PC-3 cells and ectopic ARFL-overexpressed PC-3 cells. (A) The PC-DNA cells were treated with various concentrations of R1881 as indicated for 24 h. The cells were lysed, and the MALT1 and β-actin expression was determined using immunoblot assays. The numbers indicate the ratio of target proteins/β-actin in relation to vehicle-treatment. (B) The PC-ARFL cells were treated with various concentrations of R1881 as indicated for 24 h. The cells were lysed and analyzed for AR, MALT1, NDRG1, and β-actin using immunoblot assays (top), as above. The quantitative data were expressed as the intensity of protein bands of the target proteins/β-actin relative to the control vehicle-treated group (bottom). (C) The mRNA levels of ARFL and ARv7 in the prostate cell lines, as indicated, were determined by RT-qPCR analysis (PZ: PZ-HPV-7, CA: CA-HPV-10). The mRNA levels of ARFL and ARv7 in 22Rv1 cells are regarded as 1, and ND represents non-detectable. * p < 0.05, ** p < 0.01.

Fig 4: Effects of androgen and MALT1 on PSA expression in androgen-dependent LNCaP prostate cancer cells. The mock-knockdowned LNCaP (LN_shCOL) and MALT1-knockdowned LNCaP (LN_shMALT1) cells were treated with various concentrations of R1881 as indicated for 24 h. The cells were lysed, then MALT1, PSA, and β-actin were determined by (A) immunoblot assays and (B) quantitative analysis. (C) The reporter activity of the PSA reporter vector was cotransfected with various concentrations of MALT1 expression vectors in LNCaP cells. (D) The reporter activity of the PSA reporter vector was cotransfected with MALT1 and/or AR expression vectors and treated with/without 10 nM R1881 in PCJ cells. (E) The reporter activity of the PSA reporter vector was cotransfected with MALT1 expression vectors (white bars) or control vectors (pcDNA3, black bars), and then treated with various concentrations of R1881 as indicated for 24 h in the LNCaP cells. The data are presented as mean percentages (±SE, n = 6) relative to the control vehicle-treated group. (F) The luciferase activity of the PSA reporter vector was treated with/without 10 nM R1881 and 0.1 μM MDV3100 as indicated for 24 h in LNCaP or transient overexpressed AR PCJ cells. * p < 0.05, ** p < 0.01.

Fig 5: Effect of androgen on MALT1 expression in AR-positive prostate carcer cells. The LNCaP cells were treated with various concentrations of R1881 for 24 h. The cells were lysed and then MALT1, PSA, p53, NDRG1, and β-actin were determined by immunoblot assays (A) and the quantitative data were expressed as the intensity of protein bands of the target proteins/β-actin relative to the control vehicle-treated group (B). (C) The LNCaP cells were treated with 10 nM of R1881 as indicated. The mRNA levels of MALT1 and PSA relative to time 0 were determined by RT-qPCR assays. The LNCaP were cotreated with/without 10 nM R1881 and/or 0.1 μM MDV3100 as indicated for 24 h. The cells were lysed, then MALT1, PSA, and β-actin were determined by immunoblotting (D) and quantitative analysis (E). (F) The 22Rv1 cells were treated with various concentrations of R1881 for 24 h. The cells were lysed and then MALT1, PSA, AR, and β-actin were determined by immunoblotting. The numbers indicate the ratio of target proteins/β-actin in relation to vehicle treatment. The 22Rv1 cells were cotreated with/without 10 nM R1881 and/or 0.1 μM MDV3100 as indicated for 24 h. The cells were lysed, then MALT1, PSA, and β-actin were determined by immunoblotting (G) and a quantitative analysis was presented as a relative density of MALT or PSA/β-actin (H). * p < 0.05, ** p < 0.01.