Fig 2: Regulation of the mitophagic marker PINK1 in WT and TLR4 KO mice. a PINK1 immunoreactivity was observed in the spinal dorsal horn of CCI mice. PINK1 IR cells were significantly increased in the ipsilateral compared with the contralateral side in WT mice, and no significant difference was found in TLR4 KO mice. Scale bar = 50 µm in A1, A3, A5, and A7. Scale bar = 20 µm in A2, A4, A6, and A8. b The number of PINK1 IR cells was significantly increased in the ipsilateral compared with the contralateral side of WT mice. c Expression of PINK1 was assessed by Western blotting. The PINK1 protein levels were significantly increased in the ipsilateral side compared with the contralateral side in WT mice, and no significant difference was shown in TLR4 KO mice. (D) Quantification by densitometry with Image J. Two-way ANOVA; all the data are shown as mean ± standard deviation, where *P < 0.05 denotes a significant difference compared with the control group. d Frozen sections (WT-CCI, POD7) were stained with PINK1 and co-stained with anti-GFAP (A1–4), anti-iba-1 (B1–4), and anti-NeuN antibodies (C1–4). A4, B4 and C4 is rectangular magnification of merged A3, B3 and C3, respectively. Scale bar = 50 µm in A, B, C1–3. Scale bar = 20 µm in A, B, C4

Fig 3: Impaired stabilization of PINK1 protein in AMBRA1-downregulated cells. (A-C) SH-SY5Y (A), HeLa (B) and PRKN-overexpressing HeLa (C) cells were transduced with control shRNA or shAMBRA1 and treated with CCCP as indicated (h: hours), or left untreated. PINK1 and AMBRA1 levels were analyzed by immunoblotting. ACTB or HSP90 were included as loading controls. The accompanying graphs report means ± SD of normalized PINK1 values from three independent experiments; *P < 0.05, **P < 0.01. A.U.: Arbitrary Units. Densitometric analysis of AMBRA1 levels is reported below the corresponding panels. (D) Mitochondrial fractions from control shRNA and shAMBRA1 SH-SY5Y cells were isolated as described in methods and lysed in RIPA buffer. PINK1, phospho-UBIQUITIN (Ser65) and AMBRA1 levels were analyzed by immunoblotting. OXPHOS-II SDHA and ACTB levels were included as markers of mitochondrial (M) and cytosol (C) fractions, respectively (left). The graph (right) reports means ± SD of PRKN: SDHA values of mitochondrial fractions from three independent experiments; *P < 0.05. A.U.: Arbitrary Units. (E) HeLa cells were transduced with a lentivirus encoding for PINK1-HA and overexpression monitored by immunoblotting (upper panel). PINK1-HA overexpressing HeLa cells were transduced with control shRNA or shAMBRA1 and treated with CCCP for 2 h or left untreated. PINK1 and AMBRA1 levels were analyzed by immunoblotting (middle panel). HSP90 was included as a loading control. The accompanying graph reports means ± SD of PINK1:HSP90 values from three independent experiments; ** P < 0.01, *** P < 0.001 (lower panel). A.U.: Arbitrary Units. Densitometric analysis of AMBRA1 levels is reported below the corresponding panels. (F) Control shRNA and shAMBRA1a HeLa cells were transiently transfected with LONP1a, LONP1b siRNA or a scramble control siRNA, as indicated. 48 hours after transfection, cells were treated with CCCP for 2 h. PINK1 and AMBRA1 levels were analyzed by immunoblotting. ACTB was included as a loading control (left). The graph (right) reports means ± SD of PINK1:ACTB values from three independent experiments; *P < 0.05. A.U.: Arbitrary Units. Densitometric analysis of AMBRA1 levels is reported below the corresponding panels.

Fig 4: AMBRA1 is required for an efficient activation of PINK1-PRKN signaling upon mitochondrial damage by CCCP. (A) Control shRNA, shAMBRA1a and shAMBRA1b SH-SY5Y cells (left panels) and control shRNA, shAMBRA1a and shAMBRA1b HeLa cells (right panels) were treated with CCCP for 2 h or left untreated. Phospho-UBIQUITIN (Ser65), UBIQUITIN and AMBRA1 levels were analyzed by immunoblotting. HSP90 was included as a loading control. (B) Mitochondrial fractions from control shRNA and shAMBRA1 SH-SY5Y cells were isolated as described in Methods and lysed in RIPA buffer. PRKN and AMBRA1 levels were analyzed by immunoblotting. OXPHOS-II SDHA and ACTB levels were included as markers of mitochondrial (M) and cytosol (C) fractions, respectively (upper). The graph (lower) reports means ± SD of PRKN:OXPHOS-II SDHA values of mitochondrial fractions from three independent experiments; *P < 0.05. A.U.: Arbitrary Units. (C) Control shRNA and shAMBRA1 SH-SY5Y cells were treated with CCCP for 2 h or left untreated. One hour before lysis, cells were incubated with the lysosome inhibitors bafilomycin A1, as indicated. LC3 levels were analyzed by immunoblotting (left). HSP90 was included as a loading control. The graph (right) reports means ± SD of LC3-II:HSP90 values from three independent experiments; *P < 0.05. A.U.: Arbitrary Units. (D) Control shRNA and shAMBRA1 SH-SY5Y cells were treated with CCCP for 16 h or left untreated. PARP and AMBRA1 levels were analyzed by immunoblotting. F.L.: Full Length. ACTB was included as a loading control (upper). The graph (lower) reports means ± SD of PARP CLEAVED:ACTB values from three independent experiments; ** P < 0.01. A.U.: Arbitrary Units.

Fig 5: PINK1 is responsible for CMR-induced cytoplasmic vacuolation. (a) Analysis of the effect of ectopic expression of Myc-PINK1 on CMR-induced expression levels of Alix and cytoplasmic vacuolation in LNCaP cells by Western blot (left panel), phase-contrast (middle panel) and immunocytochemistry (right panel). (b) The effect of ectopic expression of Myc-PINK1 on the expression levels of Alix in MDA-MB-231 cells as measured by Western blot. (c) The effect of siRNA-mediated knockdown of PINK1 on CMR-induced changes in the expression of Alix in MDA-MB-231 cells as analyzed by Western blot (left panel) and immunocytochemistry (right panel). Red: Calreticulin. All cells were treated with 6 µM CMR for 48 h. Cropped blots were displayed, and original images were included in Figs S21–S23.