Fig. 4

LDL supplementation rescues C4-2B and PC3 cell growth in LPDS-containing media and is blocked by inhibition of neutral cholesteryl ester hydrolysis activity. Violin plots of a NCEH1 and b LIPE mRNA expression in prostate cancer tissue compared to normal in the TCGA dataset. Violin plots of c NCEH1 and d LIPE mRNA benign, primary prostate cancer tissue and metastatic castrate-resistance prostate cancer (Met-CRPC) tissue. e Violin plots of nCEH1 protein levels in benign, primary prostate cancer tissue and metastatic castrate-resistance prostate cancer (Met-CRPC). f Representative immunoblots of neutral cholesteryl ester hydrolases hormone-sensitive lipase (HSL) and neutral cholesteryl ester hydrolase 1 (nCEH1) protein levels in C4-2B and PC3 cells. g C4-2B and PC3 neutral cholesteryl ester hydrolase (nCEH) activity of lysates from cells cultured with 1 μM of the nCEH1 inhibitor JW480 (C4-2B cells) or 1 μM JW480 and 1 μM of the HSL inhibitor 76-0079 in 10% FCS supplemented media for 2 h. h C4-2B cholesteryl ester levels following 24 h culturing in media supplemented with 10% FCS, 10% LPDS, 10% LPDS plus 50 μg/ml of human LDL (LPDS+LDL), or 10% LPDS plus 50 μg of human LDL and 1 μM of the nCEH1 inhibitor JW480 (LPDS+LDL+nCEH1i). i Pulse-Chase assessment of C4-2B cholesteryl ester turnover, where cells were either cultured for 24 h in media supplemented with 10% FCS, 10% LPDS, LPDS+LDL, or LPDS+LDL+nCEH1i, then cells cultured in LPDS+LDL or LPDS+LDL+nCEH1i were then cultured in either LPDS or LPDS+nCEH1i. j C4-2B cell proliferation cultured in media containing 10% FCS, 10% LPDS, LPDS+LDL, or LPDS+LDL+nCEH1i by IncuCyte and MTT. k PC3 cholesteryl ester levels following 24 h culturing in media supplemented with 10% FCS, 10% LPDS, 10% LPDS plus 50 μg/ml of human LDL (LPDS+LDL), or 10% LPDS plus 50 μg of human LDL and 1 μM of the nCEH1 inhibitor JW480 and 1 μM of the HSL inhibitor 76-0079 (LPDS+LDL+nCEH1i+HSLi). l Pulse-Chase assessment of PC3 cholesteryl ester turnover, where cells were either cultured for 24 h in media supplemented with 10% FCS, 10% LPDS, LPDS+LDL, or LPDS+LDL+nCEH1i, then cells cultured in LPDS+LDL or LPDS+LDL+nCEH1i+HSLi were then cultured in either LPDS or LPDS+nCEH1i+HSLi. m PC3 cell proliferation cultured in media containing 10% FCS, 10% LPDS, LPDS+LDL, or LPDS+nCEH1i+HSLi by IncuCyte and MTT. Data in a and b are represented as violin plots in GraphPad Prism: the horizontal line within the violin represents the median, and the dashed lines representing the quartiles. * P ≤ 0.05 vs. normal/benign; # P ≤ 0.05 vs. cancer by Mann-Whitney two-tailed t test (a and b) or one-way ANOVA (ce) followed by Tukey’s multiple comparisons test. Data in g–m are presented as mean ± SEM of at least three independent experiments performed in triplicate. * P ≤ 0.05 vs. FCS; $ P ≤ 0.05 vs. LPDS; † P ≤ 0.05 vs. LPDS+LDL; ‡ P ≤ 0.05 vs. LPDS+LDL+nCEH1i(+HSLi) by one-way ANOVA (g–i, k and l) or two-way ANOVA (j and m) followed by Tukey’s multiple comparisons test