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8 protocols using bafa1

1

Analyzing Lysosomal Dysfunction in Parkinson's

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In order to ensure reproducibility, the cell count was determined prior to each transfection by Cellometer® Auto T4 Plus (Nexcelom Bioscience; #SD100). SH-SY5Y CTSD KO cells were seeded into 6-well plates at 2 × 105 cells per well for western blot analysis and in same density onto 12 mm cover glasses for immunofluorescence analyses. After 24 h of expression, cells were transiently transfected with Lipofectamine 2000 (Thermo 611 Fisher Scientific; #11668027) following the manufacturer’s protocol. Cells were washed in PBS and harvested for experimental analysis after 48 h and a-syn co-transfected cells were harvested after 72 h of expression. H4 CTSD KO cells were plated at a density of 4 × 105 per well (6-well plate) and transfected 24 h later using Effectene transfection reagent (Qiagen; #301425) according to the manufacturer’s instructions. H4 CTSD KO cells were harvested after 48 h of expression. For CTSD inhibition, pepstatin A (PepA; Sigma-Aldrich; #508437) was diluted in cell culture media to a final concentration of 100 μM and incubated for 2 days after transfection. Bafilomycin A1 (BafA1) was used to inhibit the lysosome. BafA1 (Santa Cruz; #sc-201550A) was dissolved in DMSO and diluted in cell culture media to a final concentration of 0.2 μM. Cells were treated with BafA1 one day after transfection for 16 h.
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2

Embryonic Chemical Modulation

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Final concentrations of chemicals used in this study were 5 μM chloroquine (Fluka Sigma-Aldrich), 10 mM 3-MA (Sigma), 25 nM Baf A1 (Santa Cruz), 10 μM Z36 (Sigma) and 20 μM GSK2656157 (Selleck). Embryos were incubated with small molecular chemicals in embryo medium at 28.5 °C until collection.
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3

Autophagy Regulation in Cell Lines

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We used human HeLa cells (ATCC CCL-2), MEFs, Atg5 KO MEFs (Kuma et al., 2004 (link)), and CRISPR-Cas9–generated HeLa ATG7 KO cells (Mejlvang et al., 2018 (link)). All cells were cultured in DMEM (D6046; Sigma-Aldrich) supplemented with 10% FBS (S0615; Biochrom) and 1% streptomycin-penicillin (P4333; Sigma-Aldrich) and kept in a humidified incubator at 37°C and 5% CO2. Starvation experiments were conducted by incubating cells in HBSS (H9269; Sigma-Aldrich). Cells were treated with 1 µg/ml tetracycline (Sigma-Aldrich), 200 ng/ml BafA1 (sc-201550; Santa Cruz Biotechnology), and 25 µM MG132 for the indicated time periods. DNA transfection was done with Metafectene Pro (T040; Biontex) according to the manufacturer’s protocol. siRNA transfections were done with RNAiMAX according to the manufacturer’s protocol.
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4

Cellular Uptake and Cytotoxicity Assay

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Cells were seeded into a 24-well plate with MDA-MB-231 cells 25.000/well and HeLa cells 25.000/well. The protein mixtures were prepared in FCS-containing medium, 250 µL/well and added to the cells for 24 or 48 h, respectively, at 37 °C. In case of the uptake of His6-DTA, cells were pre-incubated with BafA1 (30 nM, #sc-201550, SantaCruz Biotechnology, Dallas, TX, USA) for 30 min at 37 °C. BafA1 remained present on the cells throughout the time course of the assay. Cells were washed thrice with cold PBS, the supernatant was discarded and, after a freeze–thaw cycle, lyzed in hot 2× Laemmli buffer with DTT (100 mM). The samples were subjected to SDS-PAGE and subsequent Western blotting. His6-proteins were detected with either an anti-His antibody as described above or a specific anti-NDPK-A antibody. Hsp90 was detected for comparison of equal protein loading and band intensity was quantified via the ImageJ software. For the analysis of morphological changes, the cells were monitored throughout the experiment and bright field images were acquired. The percentage of rounded cells was analyzed via blinded/masked quantification using the ImageJ software.
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5

Analyzing Membrane Trafficking Pathways

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The chemicals used in this study were: MBCD (Sigma, C4555), cholesterol-water soluble (Sigma, C4951), Baf A1 (Santa Cruz, CAS 88899-55-2), Wort (Santa Cruz, CAS 19545-26-7), cholera toxin subunit B conjugated with Alexa Fluor 594 (CTxB, Invitrogen, C34777), DAB (Dako, K401011), NEM (Sigma, E3876), DTT (Sigma, D9779), human transferrin peroxidase (Rockland antibodies and assays, 009-0334). The antibodies used were: anti-MAP1LC3B/LC3B (Sigma, L7543), anti-ACTB/β-actin (Sigma, A5441), anti-TFRC/TFRC (Invitrogen, 136,800), anti-VAMP3 (Santa Cruz, sc-514843), anti-CAV1/caveolin-1 (BD Pharmingen, 610,060), anti-ATG9A (Abcam, ab108338), anti-RAB11 (Cell Signaling Technology, 5589), mouse anti-STX6 (Invitrogen, 701,823), WIPI2 (Abcam, ab105459), and ATG16L1 (Cell Signaling Technology, 8089).
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6

Mitochondrial Dynamics Monitoring by CLEM

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Rapamycin and BafA1 were obtained from Santa Cruz Biotechnology and Focus Biomolecules, respectively. MitoTracker Green FM was purchased from Thermo Fisher Scientific. Etoposide was purchased from Sigma-Aldrich. Other chemicals, including CCCP, were purchased from Nacalai Tesque. Fluorescence images were obtained using LSM710 confocal laser-scanning microscopes (Zeiss). For CLEM observation, a JEM-1400Plus transmission electron microscope (JEOL) was used with a CCD camera (EM-14830RUBY2; JEOL).
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7

Autophagy Regulation Study Reagents

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Reagents used in this study include bafilomycin A1 (BafA1; Santa Cruz Biotechnology, sc-201,550), Hoechst 33342 (Molecular Probes/Invitrogen, H3570), torin1 (Tocris, 4247), and PMSF (Sigma, 7626).
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8

In Vitro Protein Synthesis and Inhibition

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Reagents and chemicals used were BafA1 (sc-201550; Santa Cruz Biotech), MG132 (C2211; Sigma-Aldrich), [35S]methionine (NEG709A500UC; PerkinElmer), T7-coupled reticulocyte lysate system (14610; Promega), Ponceau S (P3504; Sigma-Aldrich), DMEM (D6046; Sigma-Aldrich), bicarbonate buffered HBSS (H9269; Sigma-Aldrich), Hygromycin (10687-010; Thermo Fisher Scientific), tetracycline (87128; Sigma-Aldrich), penicillin/streptomycin (P4333; Sigma-Aldrich), Metafectene Pro (T040; Biontex), (S0615; Biochrom), Lipofectamine RNAiMAX (13778; Thermo Fisher Scientific), cOmplete EDTA-free Protease inhibitor (11836170001; Roche), chemiluminescent HRP substrate (Sigma-Aldrich), and glutathione sepharose beads (17-5132-01; GE Healthcare).
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