Fig. 6

Co-transfection of TMEM47 and MAVS or STING triggers autophagy. (A–C) Overexpression of TMEM47, MAVS, or STING could not induce autophagy. EPC cells were seeded in six-well plates and transfected with TMEM47-HA, MAVS-Myc, or STING-Myc (0.5, 1, or 1.5 µg). At 18 h post-transfection, the cells were treated with DMSO or Baf-A1 (100 nM) for 6 h. Then, the cells were harvested for IB with the indicated Abs. (D and E) Co-expression of TMEM47 with MAVS or STING specifically activates autophagy. EPC cells were seeded in six-well plates overnight and co-transfected with the indicated plasmids. At 18 h post-transfection, the cells were treated with DMSO, 3-MA (2 mM), Baf-A1 (100 nM), or CQ (100 µM) for 6 h. The cell lysates were subjected to IB with the anti-LC3, anti-Myc, anti-HA, and anti-β-actin Abs, respectively. (F and G) Co-expression of TMEM47 with MAVS or STING induces aggregation of LC3-GFP. EPC cells were plated onto coverslips in six-well plates and co-transfected with the indicated plasmids. After 24 h, the cells were fixed and observed by confocal microscopy. Earle's balanced salt solution (EBSS) was used as a positive control. Red signals represent overexpressed proteins, and green signals represent overexpressed LC3 (original magnification 63×; oil immersion objective). Scale bar, 5 µm. (H) Autophagosome-like structures detection by TEM. EPC cells were seeded in six-well plates overnight and transfected with indicated plasmids for 24 h. The cells were then analyzed by TEM, an enlarged section indicating autophagic vesicles. All experiments were repeated for at least three times with similar results.