Fig 1: Representative Western blots showing the overexpression of (A) INHBB in transfected PC3 cells and (B) INHBC in transfected PNT1A and PC3 cell lines. Graphs show fold-change of the fluorescent intensity relative to the EV control cells (dashed line at 1.0) for (C) INHBB and (D) INHBC. Data presented as individual fold-change values (dots) and the emmean fold-change ± 95% confidence limits (grey bar and lines) from 3 Western blots. ** p < 0.01 and *** p < 0.001 analysed by a NLM.
Fig 2: Representative Western blots of (A) INHBA, (B) INHBB, and (C) INHBC protein in PNT1A (PN), LNCaP (LN) and PC3 (PC) cells with GAPDH loading control. (D) Graphs showing relative fluorescent intensity of INHBA, INHBB, INHBC pro-mature multimers and INHBC mature dimer normalised to GAPDH in PNT1A, LNCaP and PC3 cells. Data is presented as the estimated marginal mean (emmean) ± 95% confidence limits. * p < 0.05, ** p < 0.01 and *** p < 0.001 relative to PNT1A cells from a General Linear Model analysis. Protein was isolated from one passage of each cell line for INHBA and INHBB and three separate passages for INHBC and quantified in three Western blots for each protein.
Fig 3: Box plots derived from gene expression data in Oncomine or FireBrowse, comparing results for human prostate expression of INHBB and INHBC. INHBB expression was shown in data sets from (A) Yu Prostate, comparing the prostate gland (P; n = 23), and prostate carcinoma (PCa; n = 65); (B) Luo Prostate, comparing benign prostatic hyperplasia (BPH; n = 9) and PCa (n = 16); (C) Tomlins Prostate data, comparing the normal prostate (P; n = 25), BPH (n = 10), prostatic intraepithelial neoplasia (PIN; n = 13), and PCa (n = 46). INHBC expression was shown in data sets from (D) Liu Prostate data, comparing prostate gland samples (P; n = 13) and PCa (n = 44); (E) The Cancer Genome Atlas (TCGA) comparing blood (n = 148), the prostate gland (P; n = 61) and PCa (n = 45). (F) FireBrowse RNASeq data comparing INHBB expression in normal prostate samples (blue; n = 52) and tumour samples (red; n = 498). (G) FireBrowse RNASeq data comparing INHBC expression in normal prostate samples (blue; n = 45) and tumour samples (red; n = 457). Figures are presented as box plots. The box midline is the median, top and bottom of the box represent 75th and 25th percentiles, and top and bottom whiskers represent 90th and 10th percentiles.
Fig 4: In vitro proliferation and migration assays for prostate cell lines either untransfected (UT), transfected with empty vector control (EV) or overexpressing INHBB or INHBC. MTS growth assay for (A) PC3 cells overexpressing INHBB, (B) PNT1A cells overexpressing INHBC and (C) PC3 cells overexpressing INHBC. Migration assays for (D) PC3 cells overexpressing INHBB, (E) PNT1A cells overexpressing INHBC and (F) PC3 cells overexpressing INHBC. Data are presented as estimated mean absorbance or cell count ± 95% confidence limits from a minimum of three separate passages with triplicate wells per assay. *** p < 0.001 analysed by NLM. ns: Insignificance is denoted by ns.
Fig 5: (A–E) MTS cell growth assays for prostate cell lines treated with exogenous recombinant INHBA, INHBB, INHBC, or INHBA or INHBB combined with INHBC (ng/mL). Data are presented as the emmean absorbance ± 95% confidence limits from a minimum of three separate biological replicates with three to six technical replicates per treatment per assay. Asterisks indicate significant differences between treated and untreated (control) cells *** p < 0.001 analysed by a NLM.
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