PHA-665752 (PHA), SU11274 (SU), Criz, Baf A1, HCQ, and MHY were purchased from Selleck Chemicals (Houston, TX). Voli was kindly provided by AstraZeneca (Cambridge, UK). HGF was purchased from Life Technologies (Frederick, MD). Reagents were formulated and stored following the manufacturer’s protocols for in vitro and in vivo experiments. Voli was formulated in a 0.5% (v/v) carboxymethylcellulose sodium solution for mice6 (link). Primary antibodies against Met, pMet (Y1234/1235), p62, LC3, mTOR, pmTOR (S2448), pULK1 (S757), Atg5, Beclin-1, NBR1, and secondary horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from Cell Signal Technology (CST, Danvers, MA). Anti-β-actin primary antibody was purchased from Sigma-Aldrich (St. Louis, MO).
Bafa1
Manufactured by Selleck Chemicals
Sourced in United States, China
BafA1 is a laboratory product that functions as an autophagy inhibitor. It disrupts the late stages of the autophagy process. The core function of BafA1 is to block the fusion of autophagosomes with lysosomes.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Selleck Chemicals (4 mentions)
Ly294002, by Selleck Chemicals (2 mentions)
Erastin, by Selleck Chemicals (2 mentions)
Z vad fmk, by Selleck Chemicals (2 mentions)
Cycloheximide (chx), by Selleck Chemicals (2 mentions)
Rapamycin, by Selleck Chemicals (2 mentions)
Bafilomycin a1, by Selleck Chemicals (2 mentions)
Lip 1, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Rapamycin, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (1 mentions)
Pha 665752, by Selleck Chemicals (1 mentions)
Anti β actin primary antibody, by Merck Group (1 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Bxpc 3, by American Type Culture Collection (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
Mg132, by Merck Group (1 mentions)
Aspc 1, by American Type Culture Collection (1 mentions)
29 protocols using bafa1
1
Investigating Autophagy Regulation in Met-Driven Cancers
2
Cell Line Culture Conditions
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All cell lines were propagated at 37°C in 5% CO2 in a humidified atmosphere. HPDE cells and HPDE-iKRAS cells were cultured as described previously (Tsang et al., 2016 (link)). AsPC-1, BxPC-3, Capan-1, Capan-2, Panc-1, and MIA PaCa-2 were purchased from the American Type Culture Collection and cultured according to the manufacturer’s recommendations. HEK293T cells were cultured in DMEM supplied with 10% FBS and 1% penicillin-streptomycin. All human cell lines were authenticated by fingerprinting using STR testing where possible and were verified to be free of mycoplasma contamination before use. In some experiments, as indicated, cells were propagated in doxycycline (100 ng/ml), 20 nM BafA1 (Selleck Chemicals), 10 µM MG132 (Sigma-Aldrich), or glucose and glutamine-free DMEM supplemented with physiologic plasma concentrations of 5 mM glucose and/or 0.5 mM L-glutamine (Corning).
3
Inhibition of Endocytic Pathways in Cells
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Cells were treated with 10 μM rabeprazole (Rabe) or omeprazole (Ome) to inhibit PPs (Selleck, Shanghai, China). Cells were treated with 10 μM Cytochalasin D to inhibit phagocytosis (MedChemExpress, Shanghai, China). Cells were treated with 10 μM indomethacin (MedChemExpress) or chlorpromazine (Selleck) to inhibit caveolae‐ or clathrin‐mediated endocytosis, respectively. Macropinocytosis was inhibited by treatment cells with 10 μM LY294002 or amiloride (Selleck). Cells were treated with 10 μM KM91104 or 10 nM Baf‐A1, or 10 μM azathioprine (Selleck) to inhibit the activity of v‐ATPase or RAC1, respectively. Cells were treated with 10 μM Dynasore (MedChemExpress), CDC42‐IN‐1 (Selleck) or NAV‐2729 (MedChemExpress) to inhibit the activity of dynamin, CDC42 and ARF6, respectively. Cell cholesterol was extracted by treatment with 10 μM MeβCD (Aladdin). Equal pH gradients in cells were induced by 10 μM 2, 4‐DNP (Merck). Cells were treated with 10 μM H89 (Selleck) to inhibit PKA activation. In most experiments, cells were pretreated with the above reagents for 2 h, followed by subsequent experiments.
4
Autophagic Flux Analysis in Huh7 Cells
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For GFP-LC3 and lamp1 colocalization, Huh7 cells were transfected with GFP-LC3 plasmid for 48 h and cells were then seeded to a cover glass slide chamber and treated with WSG, HBSS (Gibco) or Baf A1 (Selleck). Immunofluorescence staining was performed as previously described [57 (link)].
For autophagic flux analysis, Huh7 cells were seeded and grown overnight on a 20-mm-glass-bottom cell-culture dish (NEST, 801001). The formation of autolysosomes in cells treated with WSG and CQ was detected using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Thermo Fisher Scientific, P36239) according to the manufacturer's protocol. Nuclei were stained in FBS-free DMEM medium contained 5 ng/ml hoechst (Thermo Fisher scientific) at 37 °C for 10 min.
For GFP-LC3 puncta assay, cells were transfected with GFP-LC3 plasmid for 48 h, and then plated on a 20-mm-glass-bottom cell-culture dish and treated with WSG. All fluorescent images were taken using confocal microscopy (Carl Zeiss Meditec, Inc.) and quantified using Image J software.
For autophagic flux analysis, Huh7 cells were seeded and grown overnight on a 20-mm-glass-bottom cell-culture dish (NEST, 801001). The formation of autolysosomes in cells treated with WSG and CQ was detected using the Premo Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Thermo Fisher Scientific, P36239) according to the manufacturer's protocol. Nuclei were stained in FBS-free DMEM medium contained 5 ng/ml hoechst (Thermo Fisher scientific) at 37 °C for 10 min.
For GFP-LC3 puncta assay, cells were transfected with GFP-LC3 plasmid for 48 h, and then plated on a 20-mm-glass-bottom cell-culture dish and treated with WSG. All fluorescent images were taken using confocal microscopy (Carl Zeiss Meditec, Inc.) and quantified using Image J software.
5
Pharmacological Modulation of Autophagy
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L‐Ascorbic acid (Sigma‐Aldrich) was made fresh and used at a final concentration of 50 μg/ml from the beginning of each experimental procedure. bafilomycin A1 (BafA1; Sigma‐Aldrich) was used at a final concentration of 100 nM, and compared to DMSO (Sigma‐Aldrich) as vehicle for 6 h (RCS) or 9 h (Saos2/U2OS). MEFs were treated with 50 nM bafilomycin for 12 h or 100 nM for 6 h. Castanospermine (CST; Sigma‐Aldrich) was used at a final concentration of 1 mM. CST was added 2 h before BafA1, and ascorbic acid treatment. SAR405 (Selleckchem) was used at a concentration of 10 μM for 2 h preceding and throughout BafA1 treatment. Tat‐BECLIN‐1 (D17, Millipore) was used at 5 μM in acidified media for 4 h then replaced with fresh media for 2 h before harvesting cells. HaloTag, far red (ex. 650 nm, em. 668 nm) SiR HaloTag ligand (Promega), available through custom order, incubated in media at 2 mM for 3 h. 0.5 μM TMR (Promega) was added to the media 2 h pre‐fixation for lysosome visualization, or for pulse chase, 20 min at 1 μM, followed by p5030 (Promega). Rutin (Acros Organics) was used at 10 μg/ml for the duration of live cell imaging.
6
Evaluation of Cell Viability Reagents
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Erastin, RSL3, BSO, 17AAG and Baf A1 were from SelleckChem (Houston, Texas, USA). Cisplatin and 5FU were from Hospira (Lake Forrest, Illinois, USA). GSH-MEE was from Cayman Chemicals (Ann Arbor, Michigan, USA). GSH, β-mercaptoethanol, NAC, trolox, ferrostatin-1, QVD, DFO, and SAS were from Sigma-Aldrich (St. Louis, Missouri, USA). Unless otherwise specified, all general chemicals were from Sigma-Aldrich.
7
Pharmacological Modulators of Cellular Processes
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BafA1, PIK-III, and AZD8055 were purchased from Selleckchem. ML-SA1 and MK6-83 were purchased from Tocris. Monensin, nigericin, salinomycin, valinomycin, and LLoMe were purchased from Sigma-Aldrich. C8 is available for purchase through ChemShuttle (catalog no. 187417).
8
Investigating Ferroptosis Regulation in ESCC
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KYSE450 and KYSE510 ESCC cell lines, which were purchased from COBIOER BIOSCIENCE (Nanjing, China), were cultured using RPMI-1640 culture medium supplemented with 10% fetal bovine serum, streptomycin (100 mg/mL), and penicillin (100 U/mL) under conditions of 5% CO2 and 37 °C. Lipofectamine 2000 (Thermo Scientific, Carlsbad, CA, USA) was used to transfect siRNAs and vectors.
The PKCiota siRNAs, USP14 siRNA, GPX4 siRNAs, CANX siRNA, miR-145-5p mimic, NC siRNA, shPKCiota vectors, and pGCMV/EGFP/miR-145-5p vectors were synthesized by GenePharma Co., Ltd. (Shanghai, China). Information about the siRNAs is listed inTable S1 .
RSL3, Erastin, FIN56, Z-VAD-FMK, liproxstatin-1, necrostatin-1, ferrostatin-1, MG132, BafA1, IU1, and MK-2206 were obtained from Selleck Chemicals (Houston, TX, USA).
The PKCiota siRNAs, USP14 siRNA, GPX4 siRNAs, CANX siRNA, miR-145-5p mimic, NC siRNA, shPKCiota vectors, and pGCMV/EGFP/miR-145-5p vectors were synthesized by GenePharma Co., Ltd. (Shanghai, China). Information about the siRNAs is listed in
RSL3, Erastin, FIN56, Z-VAD-FMK, liproxstatin-1, necrostatin-1, ferrostatin-1, MG132, BafA1, IU1, and MK-2206 were obtained from Selleck Chemicals (Houston, TX, USA).
9
Cytokine and ISG Regulation by VSV
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BMDMs, NEs, and A549 cells were treated with or without VSV (MOI of 0.5) for the indicated time to test cytokines, ISGs expression, and signaling pathway activation. For dual luciferase assay, cells were treated as described above. CHX (100 μg/ml) (Selleck) was used to block protein synthesis, as indicated in the figures. Rapamycin (250 nM) (Selleck) or EBSS (Thermo Fisher Scientific) was used for autophagy inducement, as indicated in the figures. For protein degradation inhibition assays in HEK293T cells, 3-MA (10 mM) (Selleck) or Baf A1 (0.2 μM) (Selleck) was used to inhibit autolysosome- or lysosome-mediated protein degradation, respectively. MG132 (10 μM) (Selleck) inhibited proteasome-mediated protein degradation. Z-VAD (50 μM) (Selleck) inhibited caspase-mediated protein degradation.
10
Autophagy Flux Measurement via Baf-A1
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VSMCs were first treated with bafilomycin A1 (Baf-A1; 400 nM; Selleck Chemicals) for 4 h prior to AngII treatment as previously described, to block autophagosome-lysosome fusion. Autophagic flux was measured via western blot analysis, as previously described.
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