A combination of four siRNAs (gene accession NM_008293; FlexiTube siRNA #SI01070811, SI01070818, SI01070825, and SI01070832; Qiagen, Valencia, CA) with Lipofectimine RNAiMAX (#13778150; Life Technologies) was used to knock down 3βHSD1, according to the manufacturer’s protocol with OptiMEM reduced serum media (#31985070; Life Technologies). Optimal siRNA concentrations were determined in pilot experiments (Fig. 3B, C). For subsequent experiments, siRNA was added (75 pmol or 33 nM per well of 6-well plate, equivalent to 18.75 pmol or 8.3 nM of each of the four siRNAs). Because estradiol can induce 3βHSD1 and steroidogenic activity [35 (link),36 (link)] the cells were treated with E2 to maximize any potential 3βHSD1 activity. Briefly, 72 hours after the start of transfection, astrocytes were steroid-starved and treated with 1 nM E2 for 4 hours. Media was then collected and immediately used in conditioned media experiments or kept at −20°C until use.
Groups were analyzed using SigmaPlot for Windows version 12.5 (Systat Software, Inc., San Jose, CA). Where appropriate, Student-Newman-Keuls (SNK) post hoc tests were used (significance set to 0.05).