Tripsin edta

Control, Atg5^−/−, and Atg7^−/− MEFs purchased from Riken Cell Bank (Tsukuba, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS, Sigma-Aldrich) and 1% penicillin and streptomycin (GIBCO) at 37°C with 5% CO₂ (Kuma et al., 2004 (link)). For the adhesion assay (Hu et al., 2008 (link)), the Rho activation assay (Ren et al., 2000 (link)) and western blotting (Cheng et al., 2014 (link)), cells were grown to 60% confluence and trypsinized with 0.05% tripsin-EDTA (Gibco, Thermo Fisher Scientific, MA, USA). The trypsinization was stopped by addition of 0.5 mg/ml soybean trypsin inhibitor (Wako, Osaka, Japan) in DMEM. The cells were pelleted and washed once more with 0.5 mg/ml soybean trypsin inhibitor, followed by another wash with serum-free medium, then were suspended in DMEM containing 0.1% bovine serum albumin (BSA, Sigma) and maintained in suspension for 1 h at 37°C (Cheng et al., 2014 (link)). The suspended cells (2×10⁵ cells/ml) were plated on collagen I–coated dishes (Corning, NY, USA) and incubated at 37°C. EGFP-m-Paxillin (Plasmid #80023) was obtained from Addgene. We transfected 1 µg plasmid into 2×10⁴ MEFs using Lipofectamine® 2000 reagent (Thermo Fisher Scientific) according to the manufacturer's protocol.

Monolayer iPSC-CMs were dissociated into single cells using 0.25% Tripsin-EDTA (Gibco) for 5 min at 37 °C. Cells were pelleted and fixed with 2% paraformaldehyde (PFA) (Sangon Biotech) for 10 min on ice. Every step was washed with 5% fetal bovine serum (Gibco) in phosphate-buffered saline (PBS) (Sangon Biotech) before sample centrifugation. Cells were stained with TNNT2 (Abcam) at 4 °C. FITC-conjugated goat anti-mouse IgG antibody (Invitrogen) was used as the secondary antibody.

Monolayer CMs were dissociated into single cells using 0.25% Tripsin‐EDTA (Gibco) for 5 minutes at 37°C. Cells were pelleted and fixed with 4%PFA (Sangon Biotech) for 10 minutes on ice. Every step was washed with PBS (Sangon Biotech) before sample centrifugation. Cells were stained with TNNT2 (Abcam) at 4°C, and FITC‐conjugated goat anti‐mouse IgG antibody (Invitrogen) was used as secondary antibody.

Syngeneic Lewis Lung Carcinoma (LLC1) cells (ATCC; The Global Bioresource Center, LGC Standards, Kielpin Lomianki, Poland)—the only reproducible experimental model for the human non-small-cell lung cancer to date [46 (link),66 (link)]—were used for the induction of tumor nodules in mice. The cells were maintained as a mono-layer culture in a culture medium composed of the DMEM W/GLUTAMAX-I, PYR, 4,5 GLU medium (GIBCO, LifeTechnologies, Warsaw, Poland), 10% Heat-innactivated FBS (PAN BIOTECH, IMMUNIQ, Zory, Poland), 100 U/mL penicillin, and 100 U/mL streptomycin (Corning, Sigma-Aldrich, Poznan, Poland) in standard conditions (SC): humidified atmosphere of 95% air and 5% CO2 at 37 °C. The LLC1 cells were passaged every two days using 0.25% Tripsin-EDTA (GIBCO). The cell line was tested free of mycoplasma.