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. 2016 Oct 19;6:35531. doi: 10.1038/srep35531

Figure 4. FoxO1 regulates ATGL expression and SRA inhibits FoxO1-mediated ATGL promoter driven luciferase activity.

Figure 4

(a) Hepa1-6 cells were transfected with FoxO1 or control expression vector (pcDNA3.0). 60 h after transfection, ATGL mRNA expression (normalized to 36B4 expression) was measured by RT-qPCR (left panel) and cell extracts were immunoblotted with anti-FoxO1, anti-ATGL and anti-β-actin antibodies (right panel). (b) HepG2 cells were transfected with either pGL3-Basic empty vector (200 ng) or −3000/+ 1-LUC ATGL construct (200 ng) plus pRL-TK-Renilla (10 ng). (c) pSCT (Control, 200 ng), pSCT-SRA (SRA, 200 ng) or pSCT-SRAP-SDM1/7 (SRAP, 200 ng) were transfected with −3000/+ 1 LUC ATGL (200 ng) plus pRL-TK-Renilla (10 ng) into HepG2 cells. (d) −3000/+ 1 LUC ATGL (200 ng) plus pRL-TK-Renilla (10 ng) was transfected into HepG2 cells without or with FoxO1 (50 ng) and pSCT-SRA (SRA, 200 ng). (e) FoxO1 (50 ng) expression vector and increasing dose of pSCT-SRA as indicated were transfected with −3000/+ 1 LUC ATGL (200 ng) plus pRL-TK-Renilla (10 ng) into HepG2 cells. (f) Increasing doses of pSCT-SRA as indicated were transfected with −3000/+ 1 LUC ATGL (200 ng) plus pRL-TK-Renilla (10 ng) into HepG2 cells. Cells were treated with AS1842856 (1 μM) for 60 h after transfection. (g,h) Endogenous SRA in Hepa1-6 cells was knocked down by lentiviral infection with shRNA against SRA (shSRA) or scrambled shRNA as control (shControl). Subsequent transfection and luciferase assays were performed 12 h and 72 h after infection, respectively. (g) Hepa1-6 cells were transfected with −3000/+ 1 LUC construct (200 ng) plus pRL-TK-Renilla (10 ng) without or with FoxO1 (50 ng). (h) Hepa1-6 cells were cotransfected with −3000/+ 1 LUC ATGL (200 ng) plus pRL-TK-Renilla (10 ng) without or with AS1842856 (1 μM) treatment for 60 h after transfection. ATGL promoter driven luciferase activity were normalized to Renilla luciferase activity, presented as mean ± SE. Data are expressed as fold-change relative to the level of control. *p < 0.05.