Ab201022

Nocodazole and Ku55933 were ordered from Selleckchem (Texas, USA). Lambda Protein phosphatase (λPPase) was bought from Sigma-Aldrich Corporation. The anti-ATM (1:1,000, ab201022, Abcam), anti-phospho-ATM (1:1,000, ab81292, Abcam), anti-Mad2 (1:1,000, ab70385, Abcam), anti-Mad1 (1:1,000, ab201022, Abcam), and anti-Cdc20 antibodies (1:1,000, ab183479, Abcam) were bought from Abcam (Cambridge, MA). The anti-HA (1:1,000, 3724T, Cell Signaling Technology), anti-Mad2 (1:1,000, 4636S, Cell Signaling Technology), and the HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-Flag antibody (1:1,000, F3165, Sigma) was purchased from Sigma. The anti-Mad2 antibody (1:1,000, YT2618, Immunoway) was bought from Immunoway. The anti-GFP (1:1,000, sc-9996, Santa Cruz), anti-Mad2 (1:1,000, sc-47747, Santa Cruz), anti-p-Thr (1:1,000, sc-5267, Santa Cruz), and anti-p-Ser antibodies (1:1,000, sc-81514, Santa Cruz) were bought from Santa Cruz Biotechnology. Goat anti-mouse lgG-H HRP (1:1,000, M21004L, Abmart) and goat anti-mouse lgG-L HRP (1:1,000, M21005S, Abmart) were purchased from Abmart.

RIPA buffer (PC104, Epizyme, China) was applied to extract protein from GBM tissues followed by centrifugation. The lysed protein was quantified utilizing BCA kit (ZJ101, Epizyme, China). After electrophoresis (10% SDS-PAGE), the protein was blotted onto nitrocellulose membranes. After being sealed, the membranes were immersed in primary antibodies at 4°C overnight. A further 60 min reaction was done after the addition of secondary antibodies. After being developed with an ECL luminescence reagent (P1000, Applygen, China), the signals were analyzed by a gel imaging system. The primary antibodies of Cyclin D1 (1 : 200, ab16663), C-myc (1 : 1000, ab32072), Bad (1 : 1000, ab32445), p-p53 (1 : 5000, ab33889), ATM (1 : 1000, ab201022), Histone H2A.X (1 : 1000, ab20669), p-Histone H2A.X (1 : 1000, ab81299), phospho-STAT3 (Ser727) (1 : 1000, ab32143), phospho-STAT3 (Tyr705) (1 : 10000, ab267373), STAT3 (1 : 2000, ab68153), and GAPDH (1 : 10000, ab181602) were gained from Abcam (UK). The primary antibodies of p-AKT (1 : 2000, #4060), AKT (1 : 1000, #4685), Cleaved Caspase-9 (1 : 1000, #9507), and p-ATM (1 : 1000, #2851) were obtained from CST (USA). PD-L1 (1 : 1000, abx179111) was bought from Abbexa (USA). Pro-Caspase-9 (1 : 2000, AF6348) was bought from Affinity (USA).

Western blot analysis was performed routinely. Cells were seeded in culture plates under different conditions. All cells were washed with PBS buffer and lysed by lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were measured using the BCA method (Invitrogen Inc., United States). Equal quantities (30 μg) of proteins were loaded and separated on 10% SDS–polyacrylamide gels and then transferred onto PVDF membranes. The cells were blocked by TBST containing 5% skim milk at RT for 1 h and washed three times. The cells were incubated with primary antibodies for blots at 4°C overnight. All the primary antibodies used are listed as follows:

p-PI3K (Abcam, United States, Ab278545)

p-AKT (Abcam, United States, Ab38449)

p-mTOR (Abcam, United States, Ab109268)

GPX4 (EterLife, Birmingham, UK, EL604340)

xCT (EterLife, Birmingham, UK, EL611069)

4HNE (EterLife, Birmingham, UK, EL908789)

ATM (Abcam, United States, Ab201022)

Runx2 (Abcam, United States, Ab236639)

BMP2 (EterLife, Birmingham, UK, EL601094)

The blots were then washed in TBST and incubated with secondary antibodies at RT for 2 h. The blots were washed in TBST. The immunoreactive bands were visualized by chemiluminescence using enhanced chemiluminescence plus (GE Healthcare). GAPDH was used as an internal marker. The protein level was expressed as the ratio of the target band gray value to that of GADPH.