Journal: Neural Regeneration Research

Article Title: ATP6V0A1 protects dopaminergic neurons via the autophagy-lysosomal pathway in Parkinson’s disease

doi: 10.4103/nrr.nrr-d-24-01420

Figure Lengend Snippet: Figure 4 ｜ATP6V0A1 regulates autophagy through mTORC1 signaling. (A, B) Representative western blot bands and statistical graphs showing the levels of the autophagy-related proteins LC3 and P62 in Ctrl shRNA and ATP6V0A1 shRNA cells with or without bafilomycin stimulation for 24 hours (n = 3). (C, D) Representative western blot bands and a statistical graph of LC3 and P62 expression in control and bafilomycin- overexpressing cells with or without bafilomycin stimulation for 24 hours (n = 3). (E, F) Transmission electron microscopy images of autophagosomes in MN9D cells from the control and knockdown groups (n = 3), with the statistical graph shown in F. The arrows show autophagosomes. (G, H) Immunocytochemical staining of LAMP-1 (red, Alexa Fluor 555) and LC3 (green, Alexa Fluor 488) upon ATP6V0A1 knockdown in MN9D cells. Magnified images of LAMP-1 and LC3 staining are shown in the right column (n = 3 per group). (I, J) Representative western blot bands and the statistical graph of mTORC pathway–related proteins (mTORC, p-mTORC, S6K, and p-S6K) in Ctrl shRNA and ATP6V0A1 shRNA cells. (K, L) Representative western blot bands and a statistical graph of LC3 and P62 expression in cells subjected to rapamycin treatment for 24 hours (n = 3). All the data are expressed as the mean ± SEM. *P < 0.05, **P < 0.01. Ctrl shRNA: Lentiviral vector-transfected MN9D cells; ATP6V0A1 shRNA: lentiviral knockdown sequence-transfected MN9D cells; Vector: lentiviral vector-transfected MN9D cells; ATP6V0A1: cells constructed with ATP6V0A1 lentivirus. LAMP-1: Lysosomal-associated membrane protein 1. ATP6V0A1: ATPase H+ transporting V0 subunit A1; LAMP-1: lysosomal-associated membrane protein 1; LC3: light chain 3; mTORC1: mechanistic target of rapamycin complex 1; p-mTORC: phosphorylated mTORC; p-S6K: phosphorylated S6K; S6K: S6 kinase B1; shRNA: short hairpin RNA.

Article Snippet: Table 1 | Antibodies used in this study Antibody Dilution Cat# RRID Supplier Application Rabbit anti-β-actin 1:20000 AC026 AB_2768234 ABclonal (Wuhan, China) WB Rabbit anti-MYC-Tag 1:1000 AE010 AB_2770408 ABclonal WB Rabbit anti-ATP6V0A1 1:1000 ab105937 AB_10862106 Abcam WB Rabbit anti-LAMP-1 1:1000 ab24170 AB_775978 Abcam WB Rabbit anti-α-syn 1:1000 ab138501 AB_2537217 Abcam WB Rabbit anti-SOSTM1/p62 1:1000 39749 AB_2799160 Cell Signaling Technology (Danvers, MA, USA) WB Rabbit anti-CTSD 1:1000 2284 AB_10694258 Cell Signaling Technology WB Rabbit anti-CTSB 1:1000 31718 AB_2687580 Cell Signaling Technology WB Rabbit anti-P70 S6 kinase (S6K) 1:1000 2708 AB_390722 Cell Signaling Technology WB Rabbit anti-phospho-p70 S6K (Thr389) 1:1000 9234 AB_2269803 Cell Signaling Technology WB Rabbit anti-mTOR1 1:1000 2983 AB_2105622 Cell Signaling Technology WB Rabbit anti-phospho-mTOR (Ser2448) 1:1000 5536 Cell Signaling Technology WB Rabbit anti-p-α-syn 1:1000 23706 Cell Signaling Technology WB, IHC Mouse anti-Flag 1:1000 F3165 Sigma‒Aldrich WB Mouse anti-TH 1:1000 F-11, sc-25269 Santa Cruz Biotechnolog, Dallas, TX, USA WB, IHC Mouse anti-α-syn 1:1000 610787 BD Biosciences, San Jose, CA, USA WB Rabbit anti-microtubule-associated protein-1 LC3 II/I 1:1000 14600-1-AP Proteintech (WuHan, China) WB Rabbit anti-ATP6V0A1 1:1000 NBP1-89342 Novus Biologicals (Littleton, CO, USA) WB HRP-conjugated anti-rabbit antibody goat anti-rabbit IgG (H+L) 1:5000 65-6120 Invitrogen WB HRP-conjugated anti-mouse antibody goat anti-mouse IgG (H+L) 1:5000 62-6520 Invitrogen WB Mouse anti-LAMP-1 1:400 ab25630 AB_470708 Abcam ICC Rabbit anti-LC3A/B 1:400 12741 AB_2617131 Cell Signaling Technology ICC Alexa Fluor 488-conjugated goat anti-rabbit IgG 1:2000 4412 Cell Signaling Technology ICC Alexa Fluor 555-conjugated goat anti-mouse IgG 1:2000 4409 Cell Signaling Technology ICC Poly-HRP Anti-Mouse/Rabbit IgG Detection System GK600705A Genetech, Shanghai, China ATP6V0A1: ATPase H+ transporting V0 subunit A1; CTSB: cathepsin B; CTSD: cathepsin D; HRP: horseradish peroxidase; ICC: Immunocytochemistry; IHC: immunohistochemistry; LAMP1: lysosomal-associated membrane protein 1; LAMP-1: lysosomal-associated membrane protein 1; LC3: light chain 3; mTOR: mammalian target of rapamycin; p-α-syn: phosphorylated α-synuclein; TH: tyrosine hydroxylase; WB: western blotting; α-syn: α-synuclein.

Techniques: Western Blot, shRNA, Expressing, Control, Transmission Assay, Electron Microscopy, Knockdown, Staining, Plasmid Preparation, Transfection, Sequencing, Construct, Membrane