Fig. 4

Tamm41 regulates mitophagy progression in AC16 cells. a COS7 cells with HA-TAMM41 (red) overexpression manifest fragmented mitochondrial (COX-IV; green) appearance (white arrowhead) compared with cells without HA-TAMM41 transfection (asterisk). Scale bar: 5 μm. b Immunofluorescence assay shows diminished endogenous mitochondria (TOMM20, red) in TAMM41-GFP overexpressed 293T cells. Scale bar: 5 μm. c, d TAMM41 overexpression reduced mitochondrial protein levels in AC16 cells. e, f Mitophagy activity assessed using mt-Keima in si-control or si-TAMM41 AC16 cells. Elevated levels of mitophagy were observed following CCCP treatment in control AC16 cells, which is not detected in TAMM41 deficient ones. n = 50 cells calculated per group. Scale bar: 5 μm. g, h Western blot analysis reveal compromised CCCP-induced mitochondrial membrane protein elimination ability in TAMM41-deficient cells. i, j TEM (i) and mitochondrial contents calculation (j) show that while mitochondria engulfed by autophagosome (indicated by white arrow) and disappeared after CCCP treatment in si-control AC16 cells, enlarged mitochondria remain abundant (lower panel in i) in TAMM41 deficient AC16 cells. Black horizontal lines indicate mean ± SD. Means ± SD are shown for three independent experiments. n.s, not significant, *P < 0.05; **P < 0.01; ***P < 0.001 (Student’s t-test)