Figure 2.

Genetic Analysis of the Role of Mitochondria-Shaping Proteins in Efficiency of Pregnenolone Production during Differentiation of BeWo Cells

(A) Secretion of pregnenolone was measured at the indicated times from cells treated as shown. Where indicated, cells were fed with 22(R)-hydroxycholesterol (HCH). Data represent mean ± SEM of four independent experiments.

(B) Cells were treated with ethanol or Frk for 2 days. Where indicated, 2 hr before the collection of medium for pregnenolone measurements, cells were pretreated with DL-aminoglutethimide (AMG, 1 mM). Data represent mean ± SEM of three independent experiments.

(C) The ratio between basal and maximal pregnenolone synthesis (R_preg) determined as in (A) was calculated. Data represent mean ± SEM of four independent experiments.

(D) R_preg was determined in BeWo cells transfected as indicated and treated where indicated with Frk for 48 hr. Data represent mean ± SEM of seven independent experiments. ^∗p < 0.05 in a two-tailed Student's t test between scramble and single siRNA; #p < 0.05 in a two-tailed Student's t test between double siRNA against Mfn2 plus the indicated mitochondria-shaping protein and the corresponding single siRNA.

(E) Three-dimensional reconstructions of stacks of confocal images of lipid droplets (green) and mitochondria (red) in BeWo cells transfected with the siRNA against the indicated gene. Insets have been magnified 9×. Where indicated, cells have been treated for 2 days with Frk. Scale bar represents 20 μm.

(F) Quantification of interaction data from (E). Data represent mean ± SEM of four independent experiments.

See also Figures S1–S3.