Lc3b 2775

Primary antibodies against PLN (2D12), phosphorylated-NFATc1 (PA5-38301), total NFATc1 (MA3-024), SERCA2a (MA3-919), ryanodine receptor 1 (RyR, MA3-925), dihydropyridine receptor α1 subunit (DHPR, MA3-920), and calsequesterin I and II (CSQ, MA3-913) were obtained from Pierce Antibodies. The primary antibody directed against SLN was generated by Lampire Biological Laboratories (PA, USA) [28 (link)]. LC3B (2775) and p62 (GP62-C) antibodies were obtained from Cell Signaling Technology (MA, USA) and Progen Biotechnik (Heidelberg, Germany), respectively. The primary antibody against actin (A2066) and stabilin-2 (orb158499) were obtained from Sigma Aldrich (MO, USA), and Biorbyt (CA, USA), respectively. SERCA1a antibody (A52) was a kind gift from Dr. David MacLennan (University of Toronto) [29 (link)]. The primary antibodies against MHCI (BA-F8), MHCIIa (SC-71), and MHCIIb (BF-F3) were obtained from Developmental Studies Hybridoma Bank (IA, USA). Secondary antibodies for western blotting, goat anti-mouse IgG (peroxidase conjugated) and goat anti-rabbit IgG (peroxidase conjugated) were obtained from Santa Cruz Biotechnology (TX, USA). Secondary antibodies for immunofluorescence staining, Alexa Fluor 350 anti-mouse IgG_2b, Alexa Fluor 488 anti-mouse IgG₁, and Alexa Fluor 555 anti-mouse IgM, were obtained from Molecular Probes (OR, USA).

The following antibodies from Cell Signaling Technology were used: rabbit antibodies against LC3B (2775), P-STAT5 Y694 (9351), STAT5 (9363), P-P70S6K T421/S424 (9204), P70S6K (2708), P-ULK1 S757 (14202), P-ULK1 S638 (14205), P-ULK1 S555 (5869), ULK1 (6439), P-ATG14 S29 (92340) and ATG14 (5504). Mouse antibodies against P-AMPK T172/T183 (sc-101630) and AMPK (sc-25792) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-actin (MAB1501) was purchased from Millipore (Burlington, MA, USA). Rabbit anti-LC3B (PM036) from MBL (Woburn, MA, USA) was used for immunofluorescence analysis. Anti-rabbit and anti-mouse secondary antibodies labeled with horseradish peroxidase were purchased from Promega (Madison, WI, USA) and anti-rabbit-AlexaFluor488 was purchased from Invitrogen (Carlsbad, CA, USA). For flow cytometry analysis, the antibody anti-hCD45-V450, Annexin-V-FITC, and Cell Viability Stain from BD Biosciences (Franklin Lakes, NJ, USA) were used.

Immunofluorescence was performed 24 hrs post transfection. When necessary, 50 µM chloroquine were added for 16 hrs. The primary antibody (LC3B #2775, Cell Signaling, LabForce AG, Nunningen, Switzerland) was diluted in 1x PBS +2% NGS +0.2% Triton X-100 and incubated overnight at 4°C in a humid chamber. The secondary antibody (Alexa Fluor 594 goat α-rabbit IgG (H+L) (A11012), Molecular Probes, LubioScience, Luzern, Switzerland) was diluted in the same buffer, and incubated 1 hr at RT in a humid chamber in the dark. Nucleic acids were stained with 100 µM DAPI (4,6-diamidino-2-phenyl-indole HCl) (1/1′500 in 1x PBS) for 10 min in a humid chamber in the dark. Cells were then mounted with Citifluor AF1 (Citifluor Ltd, Leicester, UK), and conserved at 4°C. The slides were analyzed under an Olympus BX61 microscope and the Cell^M software (Olympus, Volketswil, Switzerland).

Rapamycin was purchased from MedChemExpress LLC (NJ 08540, USA). MTT, protease and phosphatase inhibitors were from Sigma-Aldrich (Oakville, Ontario, Canada). LDH-Cytotoxicity Colorimetric Assay Kit II was purchased from BioVision (Milpitas, California, USA), while Annexin V-FITC/PI Kit was from BD Bioscience. (Mississauga, ON, Canada). Autophagy Assay, Red (Cat. #9156), Intracellular Total ROS Activity Assay (Cat. #9144) and Intracellular GSH Assay (Cat. #9137) were purchased from ImmunoChemistry Technologies (Davis, CA, USA). Autophagy Inhibitor, 3-MA (Cat. #189490) and N-acetylcysteine (NAC) were from Sigma. ECL system was acquired from EMD Millipore (Billerica, MA, USA). The primary antibodies as procaspase 3 (sc-56046), procaspase 9 (sc-17784), NF-κB (sc-8008) and β-catenin (sc-59737) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), E-cadherin (8834), pERK1/2 (4370), ERK1/2 (4695), pp38 (4631), p38 (9212), cleaved caspase-3 (9664S), cleaved caspase-9 (20750S) were from Cell Signaling Technology (Danvers, MA, USA), LC3B (2775) and p62 (39,749) were all from Cell Signaling Technology (Danvers, MA, USA)and β-actin (A5441) was from Sigma-Aldrich (Oakville, ON, Canada). The secondary goat anti-mouse (554002) and anti-rabbit (554021) were from BD Pharmingen (Mississauga, ON, Canada). VersaDoc™ MP 5000 system was from Bio-Rad (Mississauga, ON, Canada).

Cells on glass coverslips were treated with either MMV652103 or vehicle for 24 and 48 h and processed for immunofluorescence with antibodies against phospho H2A.X (#2577) (1:500) or LC3B (#2775) (1:200) (Cell Signaling Technology) as previously described (Bleloch et al., 2019[8 (link)]). Cells were imaged using confocal microscopy (Carl Zeiss LSM 880 with Fast Airyscan module confocal (Oberkochen, Germany)). Multiple z layers were acquired with 1 μM step width. Images were processed using ZEN 2012 imaging software (Carl Zeiss) and maximum intensity projections were generated. For quantification, mean fluorescence was measured from at least 20 fields of view per treatment condition and pooled from three independent replicates.