C2759

Doses of 500 mg/kg 2DG (D8375, Sigma-Aldrich), 4 mg/kg CCCP (C2759, Sigma-Aldrich), 100 mg/kg meclizine (B1786, Apexbio), and 50 mg/kg or 5 µmol/kg ATP (PV3227, Thermo Fisher) were injected intraperitoneally.
We injected 500 mg/kg 2DG or 4 mg/kg CCCP immediately after surgery to investigate the numbers of surviving RGCs after inhibition of glycolysis and oxidative phosphorylation. For the analysis of glucose metabolism (Fig. 5), mice were injected with the same dose of 2DG (500 mg/kg)/iodoacetic acid (60 mg/kg)/oligomycin (0.5 mg/kg)/CCCP (4 mg/kg) 1 h prior to euthanasia.

Fibroblasts and iPSC-derived neurons were treated with 20 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP; C2759, Sigma-Aldrich) for 24 h followed by an additional treatment of PBS or NH₄Cl (15 mM) for 6 h in the presence of CCCP. After the treatment, cells were lysed in Western blot buffer and immunodetection of autophagy-involved proteins was performed as described. Quantification of autophagic synthesis was represented as the ratio between the values of the cells treated with CCCP and NH₄Cl with respect to the condition without CCCP but maintaining NH₄Cl treatment. Quantification of autophagic degradation ratio was obtained by the relation between the values of the cells treated with CCCP and NH₄Cl and the ones without NH₄Cl but maintaining CCCP treatment according to autophagy standard guidelines (Klionsky et al., 2016 (link)). See Supplementary Figure S1 for scheme of the analysis based on previous representation (Rubinsztein et al., 2009 (link)).

EtBr internalization is an indicator of envelope permeability (Ocaktan et al., 1997 (link)), whereas DHE staining reports on relative intracellular superoxide levels (Lee K. et al., 2009 (link)). Staining was carried out as described before with minor modifications (Martins et al., 2018 (link)). Where indicated, stationary phase cultures were pre-challenged with PQ or MN as described above, and without washing, cells were stained with 15 μM EtBr (Sigma #E7637) or 15 μM DHE (Thermo Fisher Scientific #D11347) for 1 h at room temperature in the dark without shaking. Since they are substrates for efflux pumps, EtBr and DHE staining was carried out in the presence of 100 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP, Sigma #C2759), a protonophore that inactivates H⁺-dependent efflux systems. Stained cells were fixed with 4% formalin (v/v) and analyzed by flow cytometry (BD Accuri C6 flow cytometer, BD Biosciences). Relative fluorescence units of individual bacterial cells were determined at Ex/Em 490/580 nm for EtBr and the DHE superoxide-reaction products (namely, 2-hydroxyethidium), and the median fluorescence intensity (MFI) of 10,000 cells was reported for each sample. To estimate superoxide levels, we calculated the MFI ratio of DHE/EtBr fluorescence in each sample to correct for probe loading (Martins et al., 2018 (link)).

JAV1 was identified as DET 021(13){IUPAC 1-[(2-amino-6-fluorophenyl)methylamino]-3-(3,4-dichlorophenoxy)propan-2-ol; SMILES NC1=CC=CC(F)=C1CNCC(O)COC=CC=C(Cl)C(Cl)=C1; May bridgecode RDR03027} and JAV2 was identified as DET 031(13) {IUPAC 3-((4-[3-chloro-5-(trifluoromethyl)-2-pyridinyl]piperazino)methyl)-1H-indole); SMILES FC(F)(F)C =CN=C(N2CCN(CC =CNC =C3C=CC=C4)CC2)C(Cl)=C1; Maybridge code HTS04862}. Both compounds were resuspended in DMSO at 20 mM and stored at room temperature. CCCP (Sigma catalog number C2759), gramicidin (Sigma catalog number G5002), polymyxin B sulfate (Sigma catalog number P1004), and polymyxin B nonapeptide (Sigma catalog number P2076) were solubilized just prior to use.

Confluent HeLa cells grown in 6‐ or 12‐well plates were treated with deferiprone (DFP; 379409, Sigma‐Aldrich; working concentration, 1 mM) for the time durations indicated. For better LC3‐II visualization, CQ (50 μM) was added to the cells to prevent lysosomal degradation for the final 12 h of DFP treatment in the experiments shown in Figs 3A and B, 4A–C, and 7A–C and in some experiments as indicated in Appendix Figs S1, S2 and S4.
Confluent HEK293 cells grown in 12‐well plates were treated with Carbonyl cyanide 3‐chlorophenylhydrazone (CCCP; C2759, Sigma‐Aldrich; working concentration 10–100 μM) for 2 h.
Confluent SH‐SY5Y cells grown in 12‐well plates were treated with Paraquat (36541, Sigma‐Aldrich; working concentration 10 μM) for 4 h.