Ms safe protease and phosphatase inhibitor

Cell pellets were resuspended in an adequate volume (approx. 2000–4000× less than the original culture volume) of 50 mM Na₂HPO₄ pH 8.0 supplemented with 1× MS-SAFE Protease and Phosphatase Inhibitor (Sigma-Aldrich, United States) and transferred to 1.5 ml reaction tubes. Cell lysis was performed by conducting a two-step sonication protocol. First, the cells were sonicated 7× 1 min in an ultrasonic bath filled with ice with short breaks in between to vortex the samples, followed by sonication with a Bioruptor UCD-200 (Diagenode, Belgium) using the following conditions: 1 min pulse and 30 s break in 10 cycles with the intensity set to “high” while cooled with ice. The extracts were cleared twice by centrifugation (21,000 × g, 20 min and 5 min, 4 °C) to remove cell debris and the protein concentration of the cleared extracts was determined by a modified Bradford assay with ROTI^®Nanoquant (Carl Roth, Germany).

Capillary Western Immunoassay was performed in protein lysates from HepG2 and HK-2 cells cultured in 6-well plates at density of 5 × 10⁵ and 1.3 × 10⁶ cells per well, respectively. After seeding, the culture medium was replaced by 2 mL of CisPt solution and the cells were treated for 6, 24 and 48 h. After treatment, the cells were washed twice with PBS 1 × and protein lysates were prepared by lysing cells with 400 µL of RIPA buffer (Sigma-Aldrich, USA) with MS-safe protease and phosphatase inhibitor (Sigma-Aldrich, USA) on ice. Capillary Western Immunoassay was performed according to manufacturer´s instructions (Protein Simple, USA). Briefly, protein lysates were analyzed on a Wes system (ProteinSimple, USA) using a 12–230 kDa Separation Module (Biotechne, UK). Levels of phosphorylated JNK (pJNK, primary antibody 1:50, Promega, USA), poly-(ADP-ribose) polymerase-1 (PARP-1; primary antibody 1:100; Cell Signalling, USA) were normalized using the reference protein β-actin (primary antibody 1:500, Sigma-Aldrich, USA). The peaks were analyzed using Compass software (Protein Simple, USA). Two criteria were used for the discrimination of signals from the background: (1) the peak high must be higher or equal to 1000 and (2) the peak’s signal-to-noise ratio given by the software must be higher or equal to 10. The results were counted as: $\frac{\text{area}\text{of}\text{the}\text{peak}\text{of}\text{interest}}{\text{area}\text{of}\text{the}\text{peak}\text{of}\mathrm{\beta }\text{- actin}}$ .

Cells were cultured up to 80% confluence in a T175 flask. Cells were washed thrice with ice-cold PBS, and the residual liquid was removed completely. Cells were lysed subsequently by adding 500 µL of lysis buffer (9.5 M urea + 2% Dithiothreitol and 1% N-Octyl-Beta-Glucopyranoside) with MS-SAFE Protease and Phosphatase Inhibitor (Sigma). The crude lysate was collected into microcentrifuge tubes and further lysed by a handheld micro-pestle. All the samples were subjected to three cycles of sonication with 5 min intervals on ice. Debris was removed by centrifugation at 10,000× g for 15 min at 4 °C. One in ten dilution of the final solution was used to determine the protein quantity using a protein assay dye kit (BioRad, Hercules, CA, USA). Samples of 50 µg of the total proteins were trypsinized overnight at 37 °C using trypsin in accordance with the manufacturer’s instruction. After trypsinizations, samples were dried using a vacuum concentrator and resuspended in 5% acetonitrile in 0.1% formic acid for mass spec analysis.