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. 2018 May 9;293(25):9724–9735. doi: 10.1074/jbc.RA118.002248

Figure 1.

Figure 1.

Knockdown of components of the DHEA sulfation pathway. A, schematic representation of the DHEA sulfation pathway. Activated sulfate in the form of PAPS is produced by either PAPSS1 or PAPSS2 and then used by the sulfotransferase SULT2A1 to convert DHEA to DHEAS. B and C, siRNA-mediated knockdown of SULT2A1, PAPSS1, or PAPSS2 in adrenocortical NCI-H295R1 cells was verified by real-time PCR and Western blotting. A scrambled oligonucleotide served as control (ctrl). Real-time PCR data normalized to 18S rRNA, fold-change relative to that control. Densitometric quantification of Western blots revealed knockdown efficiencies of up to 90% on the protein level. Double bands were interpreted as degradation products and jointly analyzed. D, DHEA sulfation was assayed for all knockdowns mentioned above, revealing functional differences between PAPSS1 and PAPSS2 for DHEA sulfation by sulfotransferase SULT2A1. Three biological replicates and their average are shown; each dot consists of at least three technical replicates. Normally distributed data were analyzed by one-way ANOVA (p value < 0.001) and post-hoc Bonferroni tests (*, p < 0.05; **, p < 0.01) relative to the control.