Chemidoc system instrument

Western blot analyses of total protein extracts were performed as previously described 6 (link). Immunodetection was performed using antibodies directed to poly (ADP-ribose) polymerase (PARP; BD Biosciences San Diego, CA, USA), LC3B (Cell Signaling Technology, Danvers, MA), HSP72/73 (Calbiochem, EMD Biosciences, La Jolla, CA), Bcl-2 (SC-509, Santa Cruz Biotechnology Inc, Dallas, TX, USA), Bcl-xL (SC-271121, Santa Cruz Biotechnology Inc), Mcl-1 (SC-12756, Santa Cruz Biotechnology Inc) α-tubulin (Santa Cruz Biotechnology Inc) actin (Sigma-Aldrich, St. Louis, Missouri, USA). Anti-rabbit or anti-mouse immunoglobulin G-horseradish peroxidase-conjugated antibodies (Amersham Biosciences, Freiburg, Germany) were used as secondary antibody. Images were acquired by Image Lab Software (Bio-Rad, Hercules, CA, USA), using a ChemiDoc System instrument (Bio-Rad). The densitometric evaluation was performed using Image J software and normalized with relative controls.

Cells were lysed in RIPA buffer in presence of protease and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentrations were determined by colorimetric assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific). Western blotting was performed using 35–40 µg of protein extracts (60 µg for the detection of LATS1 phosphorylation), using the following primary antibodies: α-tubulin (sc- 32293), Bcl-2 (sc-509) and Bcl-xL (sc-8392) were from Santa Cruz Biotechnology YAP (#12395), phosphorylated YAP (S127, #4911), LATS1 (#3477), phosphorylated LATS1 (#8654), MST2 (#3952), MOB1 (#13730), CTGF (connective tissue growth factor, also known as CCN2, #10,095), vinculin (#13901), pERK1/2 (#9106), ERK (#9102) and H3 (#4499) were from Cell Signaling (Danvers, MA, USA); β-actin (#A1978), alpha-smooth muscle actin, α-SMA (#A5228) was from Sigma-Aldrich; heat shock protein (HSP)72/73 (#HSP01) was from Calbiochem (San Diego, CA, USA). Enhanced Chemiluminescent Substrate method (LiteAblotTURBO, Euroclone) was used to detect immunostained bands, except for the detection of phosphorylated LATS1, by Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA). ChemiDoc System instrument (Bio-Rad Laboratories) was used to acquire images, while ImageJ software was used for densitometric evaluation and normalization with relative controls.

Cells were lysed in 10 mmol/L trisaminomethane hydrochloride buffer pH 7.4 with
2% sodium dodecyl sulphate (SDS) and fresh protease inhibitors. Protein
concentrations were determined by colorimetric assay after extracts sonication
for 20 s (Pierce™ BCA Protein Assay Kit, Thermo Scientific, Waltham,
Massachusetts, USA). The following primary antibodies were used to perform
western blotting: Bcl2L10 (#3869, Cell Signaling, Danvers, MA, USA),
p44/42 [extracellular-signal-regulated kinase (ERK)1/2, #9102,
Cell Signaling], phosphorylated p44/42 (ERK1/2, #9106 L, Cell
Signaling) and MMP2 (H-76, sc-10736, Santa Cruz Biotechnology, Santa Cruz, CA).
β-actin (#A1978, Sigma-Aldrich) and heat shock protein
(HSP)72/73 (#HSP01, Calbiochem, San Diego, CA, USA) were used to check
equivalent transfer and loading. Chemiluminescent method (Pierce, Rockford, IL)
was used to detect immunostained bands. Image Lab™ Software (Bio-Rad,
Hercules, CA, USA) and ChemiDoc System instrument (Bio-Rad), were used to
acquire images, while ImageJ software was used for densitometric evaluation and
normalization with relative controls.
Cultured medium (CM) from M14-B and M14-C cells incubated in serum free medium
(SFM) for 24 h was normalized to the number of adherent cells and assayed for
gelatinase activity using 7.5% SDS gels containing gelatin (0.1 mg/mL)
as previously described [31 (link),
32 (link)].