Flotillin-1 promotes the progression of hepatocellular carcinoma by activating TFE3-mediated Golgi stress response via inhibition of mTORC1/2

Figure 4 Flotillin-1 promotes the dephosphorylation and nuclear translocation of Golgi stress transcriptional regulator transcription factor E3 by recruiting mechanistic target of rapamycin complex 1/2. A-F: Flotillin-1 (FLOT1) overexpression was transfected in Huh-7 cells and FLOT1 short-hairpin RNA was transfected in SNU-387 cells (n = 3). Expression of total protein FLOT1 and phosphorylation of transcription factor E3 (TFE3) protein Ser321 (A) was detected by western blot. Expression of TFE3 in the nucleus (B) and cytoplasm (C) was detected by western blot. Expressions of FLOT1 (D) and TFE3 (E) mRNA were detected by quantitative reverse transcription polymerase chain reaction with GAPDH as a reference. Immunofluorescence double staining of FLOT1 and TFE3 expression and localization. Scale bar = 50 μm (F). Values are expressed as mean ± SD (n = 3), and comparisons between two groups were performed using the t-test. ^aP < 0.05, ^bP < 0.01; G: Reporter vector GASE_LUC was constructed, and 293T cells were transfected with OV-TFE3 and OV-FLOT1, respectively. After 48 hours of transfection, the dual luciferase reporter assay was performed; H-J: SNU-387 cells were transfected with short-hairpin RNA-FLOT1 and treated with the mechanistic target of rapamycin complex 1/2 inhibitor Torin1. Phosphorylation status of TFE3 Ser321 (H) was assessed via western blot analysis. Additionally, the expression levels of TFE3 in both the nucleus (I) and cytoplasm (J) were evaluated using western blot. Values are expressed as mean ± SD (n = 3), comparisons among multiple groups, one-way ANOVA was employed, followed by LSD test or Tamhane’s T2 test. ^aP < 0.05, ^bP < 0.01. FLOT1: Flotillin-1; OV: Overexpression; TFE3: Transcription factor E3; shRNA: Short-hairpin RNA.