Fig. 5: TFEB overexpression restores the STB phenotype in TFEB KO cells.

A Gene-set Enrichment Analysis was performed on genes ranked by their fold-change and significance (see “Methods”) in TFEB knock-out cells transduced with a TFEB overexpressing vector (KO + TFEB) against an empty control vector (KO + EMPTY), upon treatment with Forskolin (FRSK - left) or Torin-1 (TORIN - right) in RNAseq (upper panels) and LC–MS/MS (lower panels). Upregulated genes from panel S1A (STB signature - BeWo) were used as geneset. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are reported. B Representative image of immunoblot analysis of CYP19A1 and TFEB levels in TFEB knock-out cells transduced with an empty control vector (KO + EMPTY) or TFEB overexpressing vector (KO + TFEB) upon treatment with DMSO, Forskolin (FRSK) and Torin-1 (TORIN). GAPDH was used as a loading control. C Bar chart graph representations of the quantitative determination of 17β-Estradiol (E2) and Estrone (E1) in TFEB knock-out cells transduced with an empty control vector (KO + EMPTY) or TFEB overexpressing vector (KO + TFEB) upon treatment with DMSO, Forskolin (FRSK) and Torin-1 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).