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. 2016 Sep 13;101(12):4585–4593. doi: 10.1210/jc.2016-2864

Figure 4.

Figure 4.

Adrenocortical cell knockdown of CYB5A. A, Expression of green fluorescent protein in HAC15 adrenocortical cells transduced with lentiviral shRNA. Scale bars, 200 μm. B, Knockdown of CYB5A mRNA in scrambled and CYB5A-KO cells. Data are shown as the percentage change compared with scrambled. Results are given as mean ± SEM. Statistical analyses were performed using the t test, *P < .001 vs scrambled. C, Immunocytochemistry denoting expression of CYB5A protein (red) in scrambled and knockdown cells. Nuclei are counterstained with DAPI (blue). D, Effect of CYB5A knockdown on HAC15 cell production of Δ5-steroid sulfates. Scrambled and CYB5A-KO cells were treated with the 10 μM forskolin for 48 h. The Δ5-steroid sulfate content of media was determined using LC-MS/MS and normalized with cellular protein content. Results represent the mean ± SEM from three independent experiments performed in triplicate. Statistical significance was determined using a one-way ANOVA followed by Holm-Sidak test. *, P < .05 vs scrambled under basal conditions; $, P < .05 vs scrambled under forskolin-stimulated conditions; #, P < .05 vs basal in the same cell type. E, Ratios of DHEA-S/17OHPreg-S in scrambled and CYB5A-KO. Scrambled and CYB5A-KO cells were treated with the 10 μM forskolin for 48 h. The Δ5-steroid sulfate content of media was determined using LC-MS/MS and normalized with cellular protein content. Ratios of DHEA-S/17OHPreg-S were calculated and compared in different conditions. Results represent the mean ± SEM from three independent experiments performed in triplicate. Statistical significance was determined using a one-way ANOVA followed by Holm-Sidak test. *, P < .05 vs scrambled under basal conditions; $, P < .05 vs scrambled under forskolin-stimulated conditions; #, P < .05 vs basal in the same cell type.