Ab169197

GSK-J4, a UTX-specific pharmacological inhibitor (SML0701, Sigma-Aldrich, St. Louis, Missouri, USA), reconstituted in DMSO, with different concentrations (0, 2, 4, 8 µM) were given to these cells for 48 h. Whole-cell lysates of GSK-J4 treated cells were extracted using the fixture of RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS)) and subjected to western blot analyses. The membranes were incubated with primary antibodies against JMJD3 (ab169197, 1:2000, Abcam, Cambridge, MA, USA), Rb (#9309, 1:1000, cell signaling, Danvers, Massachusetts, USA), phosphorylated Rb (pRb) (ab173289, 1:1000, Abcam, Cambridge, UK), p21 (#2947, 1:1000, Cell Signaling, Danvers, Massachusetts, USA) and β-actin (A5441, 1:10000, Sigma-Aldrich, St. Louis, Missouri, USA) at 4 °C overnight. After that, the membranes were incubated with secondary antibodies for one hour at room temperature to reveal the immune detection. The protein signals were detected with Western Lightning Chemiluminescence Reagent Plus (Amersham Biosciences). All the experiments were repeated at least three times with similar results.

Tissue or cells samples were lysed with ice‐cold RIPA buffer with complete protease and phosphatase inhibitors. The protein concentrations were measured using BCA protein assay kit. Total proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies: AMPK (1:1000, ab169197, abcam) and p‐AMPK (1:1000, 3934, Cell Signaling Technology, Inc.), TRIM11 (1:1000, ab191217, abcam), GPX4 (1:1000, ab267373, abcam), and β‐Actin (1:5000, Santa Cruz Biotechnology) after blocking with 5% BSA in TBS, followed by incubation with peroxidase‐conjugated secondary antibodies (Santa Cruz Biotechnology). The signals were detected with the ECL system and exposed by the ChemiDoc XRS system with Image Labsoftware (Bio‐rad).

Cell lysates were obtained by extraction with protein lysis buffer supplemented with protease inhibitors. Lysates were then resolved using SDS-PAGE. The protein gel is then transferred to a PVDF membrane and closed with 5% skim milk powder and incubated overnight at 4 °C with the primary antibody. The PVDF membrane is then incubated with the appropriate secondary antibody. Signals were analyzed using ECL detection reagents (Millipore, St. Louis, MO, USA). The antibodies used in this study are as follows: anti-KDM6B (ab169197, Abcam), anti-β-actin (60008-1, Proteintech, Wuhan, PR China), anti-CXCR4 (ab181020, Abcam), anti-E-Cadhrein (3195, Cell Signaling Technology), anti-N-Cadhrein (13116, Cell Signaling Technology), anti-AKT (4691, Cell Signaling Technology), anti-p-AKT (4064, Cell Signaling Technology), anti-ERK (4695, Cell Signaling Technology), and anti-p-ERK (4370, Cell Signaling Technology).