Fig. 5. Risk allele increases prostate cancer risk by gain of SPDEF.

a Position weight matrix analysis shows a gain of strong motif of SPDEF in the risk allele “T” of rs72725854. b EMSA showing differential affinities of the risk “T” and non-risk “A” alleles of rs72725854 for SPDEF. The experiment was performed thrice. c The graph depicting FLAG-SPDEF ChIP-qPCRs on plasmids harboring different alleles of rs72725854, PCAT1 intron, PVT1 promoter, and two negative controls in LNCaP cells overexpressing 3xFLAG-SPDEF. d Immunoblot with anti-SPDEF, FLAG, and GAPDH antibodies showing the levels of SPDEF and GAPDH proteins upon overexpression and the knockdown of the SPDEF protein by 3xFLAG-SPDEF and on-target siRNA pools, respectively. The experiment was performed thrice. e Reporter assays show the alterations in activities of non- risk and risk alleles upon specific knockdown of SPDEF. f Reporter assays show the alterations in reporter activities of non-risk and risk alleles upon SPDEF overexpression. g Reporter assays show the change in the luciferase activity in a dose-dependent manner when the percentage of the plasmid with the “T” allele increases over “A” allele in the pool of “A” and “T” alleles. h Competition reporter assays show the alterations in reporter activity of “T allele-Luc” upon dose-dependent (0, 50, 150, 250, 350 ng) overexpression of “A (Non-Luc)” or “T(Non-Luc)” plasmids. i Graph depicting qPCR signals on the “A” and “T” plasmids in DNase I hypersensitivity assays performed in LNCaP cells (n = 2). Error bars denote SEM from three biological replicates in c, n > 3 replicates in e–h. p-values were calculated by Student’s two-tailed unpaired t-test in c, e, f and g. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ^nsp > 0.05. Source data are provided as a Source Data file.