Fig. 5

Model of proteins involved in steroid hormone synthesis, identified proteins in proteome of BeWo cells and calcium dependence of FDX and HSD11B2. (A) General schematic overview of steroidogenic pathways occurring in human tissues with enzymes involved in steroid hormone synthesis and potential intermediates. Mass spectrometrical identified proteins in BeWo cells are highlighted with filled boxes and white letters: in grey, proteins that were identified but not differentially expressed in a FSK or calcium-dependent manner, in blue, proteins that were upregulated in a calcium-independent manner after FSK stimulation and in red, proteins that were upregulated in a calcium-dependent manner after FSK stimulation in BeWo cells. (B) Expression analyzes of aromatase (CYP19A1). Top left, western blot of BeWo RIPA lysates incubated with antibodies directed to aromatase (top) and calnexin (bottom). Cells were cultured in the presence of normal calcium (lane 1), normal calcium + FSK (lane 2), low calcium (lane 3), low calcium + FSK. Top right, statistical analysis of 4 western blots normalized to calnexin. Aromatase amount, of cells grown in the presence of normal calcium was set to 100% (factor 1). One way ANOVA test, multiple comparison with Bonferroni, p < 0.05, N = 4. Bottoms left/right, MS analysis of aromatase (CYP19A1) abundance in BeWo cell lysates (N = 4) cultured in the presence of normal/low calcium +/- FSK, unpaired t-test, N = 4 (C-D) MS analysis of several dysregulated proteins involved in synthesis of steroid hormones in the presence of different calcium-concentrations (low L and normal N) +/- FSK, Adrenodoxin FDX (C, top), HSD11B2 (C, bottom), HSD17B1 (D, top) and CYP11A1 (D, bottom), N = 4, unpaired t-test (E) Western blot of lysates incubated with HSD11B2 antibody in the presence of normal calcium (lane 1), normal calcium + FSK (lane 2), low calcium (3), low calcium + FSK (lane 4) (E, top) and calnexin control (E, bottom)