P16ink4a

The cultured cells were solubilized in lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris–HCl, 5 mmol/L EDTA–2Na, 1% Triton X-100, and 1 tablet/10 mL complete mini EDTA-free) and centrifuged at 15,000 × g at 4 °C for 30 min. Samples were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were first blocked in a buffer containing 25 mmol/L Tris–HCl (pH 7.4), 150 mmol/L NaCl, 0.1% Tween 20, and 4% skim milk for 1 h and then incubated with primary antibodies at 4 °C overnight. This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h. Primary antibodies against human p21^Waf1/Cip1, p16^INK4a, phosphorylated H2AX (Ser139, γ-H2AX), retinoblastoma (Rb), phosphorylated Rb (Ser780, pRb), cyclin D, and caspase-3 were all purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Antibodies against human cyclin A (Novocastra Laboratories Ltd., Newcastle, UK), p16^INK4a (BD Biosciences, Inc., Farmingdale, NY, USA), Ki-67 (Dako from Agilent, Santa Clara, CA, USA), p62/SQSTM1, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were also used. The immunoreactive proteins were then detected by enhanced chemiluminescence (GE Healthcare, Fairfield, CT, USA). Immunoblots were quantified using the CS Analyzer 3.0 software (ATTO, Tokyo); β-actin expression was used as the internal control.

Proteins were extracted from cell monolayers as described previously (Gorwood et al., 2019 (link); Gorwood et al., 2020 (link)). After SDS-PAGE, the proteins were transferred to nitrocellulose membranes. Specific proteins were detected using antibodies against p16^INK4A, p21^WAF1 (BD Bioscience, Franklin Lakes, NJ), prelamin A, PPARγ, CEBPα, SREBP1c, AMPK, phospho-AMPK (Cell Signaling Technology, Danvers, MA), and the protein loading control tubulin (Merck, Sigma-Aldrich). Immunoreactive complexes were detected using HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) and enhanced chemiluminescence (Thermo Fisher Scientific).

Five μm sections were cut from paraffin blocks and mounted on charged slides. They were deparaffinized in xylene and rehydrated through graded concentration of ethanol. Following that, they were immersed in citrate buffer pH 6.0 at 95–101 degrees C for 20 minutes. Once the slides were cooled down they were rinsed & placed in Tris buffered saline. Endogenous peroxidase is quenched with 3% Hydrogen peroxide. The primary antibody p16^ink4a (BD Biosciences, catalog # 551154. San Jose, CA) was applied at a dilution of 1:200. The antibody was detected with Envision+ from DAKO (catalog# K4001. Carpentaria, CA) then visualized with Diaminobenzidine and counterstained with hematoxylin. Slides were then dehydrated, cleared & mounted with resinous mounting media.