Fig. 4: TFEB drives STB endocrine functions.

A Analysis of secreted proteins through LC–MS/MS in wild-type (WT) and TFEB knock-out (KO) cells. (Left) Pie chart showing the percentage of detected proteins classified into “not secreted” (92.83%) and “secreted” (7.17%) according to Human Protein Atlas. (Right) Bar plot showing the number of secreted proteins (x-axis) belonging to the indicated Biological Process categories (y-axis). B (Left) Volcano plot showing secreted proteins displayed as Log₂ Fold Change in wild-type (WT) cells upon Forskolin (FRSK) treatment compared to DMSO conditions - FRSK vs DMSO (WT) on the x-axis - and in TFEB knock-out (KO) cells treated with FRSK with respect to WT in the same conditions - KO vs WT (FRSK) on the y-axis. Proteins upregulated upon FRSK treatment in WT cells and downregulated in KO versus WT cells in FRSK conditions are represented as light blue dots. (Right) Table displaying selected proteins and their corresponding Log₂ Fold Change in indicated conditions. C Schematic representation of the estrogen biosynthetic pathways from cholesterol to 17β-Estradiol (E2). Major enzymes synthesizing indicated steroid intermediates are displayed in red (directly bound by TFEB from our ChiPseq analysis) and in blue (not directly bound by TFEB but affected by its downregulation). D Representative image of immunoblot analysis of CYP19A1 levels in cells wild-type (WT) and TFEB knock-out cells (KO#1 and #2) in DMSO e Forskolin (FRSK) conditions. GAPDH was used as a loading control. E Bar chart graph representations of the quantitative determination of 17β-Estradiol (E2) and Estrone (E1) in wild-type (WT) and TFEB knock-out (KO) cells in DMSO and Forskolin (FRSK) conditions detected by UHPLC–MS/MS-based targeted steroidomics. Steroid concentration values are expressed as ng/mg of protein. Statistical analysis was performed by One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).