*2.2. Cell Culture and Cell Viability*

The LLC-PK1 cells were cultured in DMEM medium, supplemented with 10% FBS and penicillin/streptomycin (100/50 U/mL) in a humidified incubator with 5% CO2 atmosphere at 37 ◦C. The experiments were carried out at a density of 50,000 cell/cm2, passaged 9 to 30 times since the acquired cryovial was opened. The cells were grown in 100 mm culture dishes and when they reached 90% confluence, were planted in the corresponding experimental plates. After 24 h of growth in 10% FBS medium, LLC-PK1 cells were exposed to 0 to 5 μM αM, 5 to 40 μM CDDP or co-incubation of αM and CDDP for 24 h in 1% FBS medium. Stock 10 mM αM was dissolved in DMSO and a posterior dilution of 1:100 in phosphate buffer saline (PBS) (100 μM) was used to prepare each experimental concentration. A stock 0.5 mg/mL (1.670 mM) of CDDP in 0.9% saline solution was used for different CDDP concentrations. Additional treatment consisted of pre-treatment with 30 μM CQ (2 h) or 10 nM wortmannin (1 h previous and during different treatment: αM, CDDP or αM+CDDP) as autophagy inhibitors. After pre-treatment, αM and/or CDDP were added for 24 h. One bright field image was captured in triplicate with a 20× objective after treatments, using a Cytation 5 Cell Imaging Multi-Mode reader (BioTek Instruments, Winooski, VT, USA).

Cell viability was measured by the FDA assay according to a previously described method [26] in 96-well tissue culture microplates, where fluorescence is directly proportional to cell viability.
