Usp39

Protein extraction and western blot assay were performed as previously described [36 (link),37 (link)]. Antibodies used are as follows: USP39 (1:2000, Abcam, Cambridge, UK), p53 (DO-1, 1:300, Santa Cruz, CA, USA); p21(12D1, 1:2000, CST); CDC2 (1:1000, Ruiying Biological, Suzhou, China); CyclinB1 (1:1000, Abcam, UK); MMP2 (1:1000, Ruiying Biological, Suzhou, China); MMP9 (1:1000, Ruiying Biological, Suzhou, China); cleaved cas3 and cleaved cas9(1: 1000, Proteintech, Wuhan, China), 53BP1and γH2AX (1:2000, Abcam, UK), p-p53(s15) (1:1000, ABclonal, Woburn, MA, USA), CDK2 (1:1000, Proteintech), CyclinA2 (1:1000, Proteintech), BAX (1:1000, Proteintech), and β-actin (1:40000, Sigma, St. Louis, MO, USA). Additionally, cycloheximide (CHX) and pifithrin-α(PFT-α) were purchased from MedChemExpress.

Cells and tissues were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing a protease inhibitor cocktail (Sigma-Aldrich). Protein lysates (20 μg) were run on SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% skim milk, and incubated overnight with primary antibodies at 4 °C followed by incubation with secondary antibodies (ZSGB-BIO). Proteins on membranes were visualized using the Chemiluminescent Reagents Kit (Millipore; Billerica, MA, USA). Signals were detected with the Chemi-Doc XRS+ (Bio-Rad; Hercules, CA, USA) and quantified using Image Lab 3.0 software (Bio-Rad). The following primary antibodies were used for western blotting: USP39 (#ab131332, Abcam), Histone H3(#ab176842, Abcam), Ki67 (#ab92742, Abcam), GAPDH (#sc-25778, Santa Cruz Biotechnology; Dallas, TX, USA), LATS1(#3477, Cell Signaling Technology), LATS2(#5888, Cell Signaling Technology), YAP (#14074, Cell Signaling Technology), and TAZ (#8418, Cell Signaling Technology).

Lysates from cells and tissues were prepared using RIPA buffer containing a protease inhibitor cocktail (Sigma‐Aldrich, Merck, kGaA, Darmstadt, Germany). Proteins were separated using SDS/PAGE gels and were electrotransferred to nitrocellulose filter membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% non‐fat milk and immunoblotted with primary specific antibodies overnight at 4 °C, followed by their respective secondary antibodies. The primary antibodies were used as follows: ADAM9 (#ab186833; Abcam, Cambridge, MA, USA), USP39 (#ab131244; Abcam), integrin β1 (#34971; CST, Danvers, MA, USA), GAPDH (#60004‐1‐Ig; Proteintech, Wuhan, China), β‐Actin (#AA128; Hua An Biotechnology, Hangzhou, China).