Figure 4.

XBP1s-mediated control of mitophagy is fully PINK1-dependent. (A-L) Control (PINK1 CT) or PINK1 knocked-down (PINK1 KD) SH-SY5Y cells were transiently transfected with an empty vector (Ev) or with Xbp1s (Xbp1s) cDNA. Twenty-four hours after transfection, BECN1 (A and B, N = 8) LC3-II:LC3-I ratio (A and C, N = 8), SQSTM1 (A and D, N = 8), OPTN (A and E, N = 8), CALCOCO2 (A and F, N = 8), TIMM23 (A and G, N = 12), TOMM20 (A and H, N = 12), HSPD1 (A and I, N = 12), UB (S65) (A and J, N = 8), PRKN (A and K, N = 8) and MFN2 (A and L, N = 12) protein expressions were analyzed by western blot as described in Methods. ACTB expression is provided in (A) as a control of protein load. (M) PINK1 CT and PINK1 KD cells either empty vector (-) or Xbp1s (+) cDNA-transfected were analyzed by flow cytometry to measure mitochondrial membrane potential as described in Methods. (B-M) Data are expressed as percent of PINK1 CT non-transfected cells (taken as 100%) and are the means ± SEM. of 4–6 independent experiments performed in duplicates. Statistical analyses were performed by Kruskal Wallis multiple comparison test (B) and Sidak’s multiple comparisons test (C-M). Statistical significances: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001