Cell lytic

Protein isolation from cells was conducted using Cell Lytic (Sigma-Aldrich, St. Louis, MO, USA) with cOmplete™, Mini Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Protein isolation from explants was carried out using T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Isolated protein from cells and placental explants was quantified using BCA Assay concentrate (Bio-Rad Laboratories, Foster City, CA, USA) with BSA as standard.

Total protein was harvested from asynchronously proliferating cells in lysis buffer (CellLytic, Sigma-Aldrich) supplemented with ULTA protease inhibitors and PhosSTOP phosphatase inhibitors (Roche Diagnostics, UK). Lysates were sonicated, the supernatant was collected, and protein concentrations were quantified with a BCA Protein Assay kit (Pierce, Rockford, IL, USA). Cell lysates were resolved on 10% SDS-PAGE gels and transferred to 0.2 μm PVDF membranes by western blotting. The membranes were blocked with 5% nonfat dry milk at room temperature for 1 h and then incubated overnight with the following specific primary antibodies: anti-caspase 3 (#9662), anti-PARP (#9542), anti-Survivin (#2803), anti-nucleophosmin (#3542), anti-nucleophosmin phospho-Thr199 (#3541), anti-GAPDH (#2118), and anti-β-actin (#5125) were from Cell Signaling Technology; anti-retinoblastoma phospho-Ser807/801 (#sc-819), anti-MCL-1 (#sc-819), anti-p53 (#sc-126), and anti-Cdc2 (#sc-54) were from Santa Cruz Biotechnology; and anti-RNA polymerase II CTD repeat YSPTSPS Phospho-Ser2 (ab70324) was from Abcam.

The hWJSC extracts, namely, the hWJSC-conditioned medium (hWJSC-CM) and the hWJSC-lysate (hWJSC-L), were prepared according to an earlier published protocol (Gauthaman et al., 2012 (link)). Briefly, the hWJSC-CM was prepared from the early passages (P3–P4) of the hWJSCs (70–80% confluence) cultured in the hWJSCs medium for 48 h under standard culture conditions of 37°C and air atmosphere of 5% CO₂. The medium was separated, filtered through a 0.22 mm Millex-GP syringe filter (Millipore, MA, United States), and stored at −20°C until use in experiments. The hWJSC-L was prepared by lysing the pelleted hWJSCs using 100 μl commercial mammalian cell lysis buffer (Cell Lytic, Sigma, MO, United States) and incubation on ice for 45 min. The lysate was centrifuged (15,000 rpm, 15 min), and aliquots of the supernatant were stored at −80°C until use in experiments. The total protein content of the hWJSC-L was measured using a Nanodrop^TM spectrophotometer (Nanodrop Technologies, DW, United States).

A total of 1–3 × 10⁴ cells were lysed using CellLytic (Sigma-Aldrich). We used an iBlot kit (Invitrogen) to perform immunoblotting. The antibodies used to detect proteins were as follows: Anti-SLAMF8 antibody (rabbit polyclonal, PAB8582; Abnova, Heidelberg, Germany), anti-phospho-ALK (Tyr 1096) (rabbit polyclonal, #4143; Cell Signaling Technology, Beverly, MA, USA) and anti-GAPDH antibody (rabbit monoclonal [D16H11], #5174; Cell Signaling Technology). The blotting images were scanned using a Light-Capture II (ATTO) (Figs. 1 suppl and 2 suppl). To quantify the band’s intensities, the scanned data were analyzed using the CS Analyzer (ATTO).

Collagenase was obtained from Worthington Biochemical Corporation (Labclinics, Madrid, Spain). Cell Lytic for cell lysis and protein solubilization, crystal violet, protease inhibitor cocktail (Complete, EDTA-free), thapsigargin and Tween-20 were purchased from Sigma Chemicals Co. (Madrid, Spain). Fetal bovine serum, Hank’s balanced salts (HBSS), horse serum, medium 199 and SuperSignal West Femto were obtained from Fisher Scientific Inc. (Madrid, Spain). Polystyrene plates for cell culture were obtained from Thermo Fisher Sci. (Madrid, Spain). Penicillin/streptomycin was purchased from BioWhittaker (Lonza, Basel, Switzerland). Acrylamide, Bradford´s reagent, Tris/glycine/SDS buffer (10×) and Tris/glycine buffer (10×) were from Bio-Rad (Madrid, Spain). 5-Bromo-2-deoxyuridine (BrdU) cell proliferation assay kit was purchased from BioVision (Deltaclon S.L., Madrid, Spain). SB203580 and U0126 were obtained from Tocris (Biogen Científica, Madrid, Spain). Species-specific HRP-conjugated secondary antibodies were purchased from Thermo Fisher Sci. (Madrid, Spain). All other analytical-grade chemicals used were obtained from Sigma Chemicals Co. (Madrid, Spain).