Becn1

Metformin was purchased from Sigma-Aldrich. The primary antibodies of GAPDH, BECN1, LC3, BNIP3, BCL2 and E2F1 were acquired from Proteintech (Wuhan, China). The 4, 6-diamidino-2-phenylindole (DAPI) was purchased from Solarbio (Beijing, China). The receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) were obtained from R&D Systems (Minnesota, USA). We purchased Minimum Essential Medium Alpha (MEM-α), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin from Gibco (Grand Island, NY, USA). The cell counting kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). Enzyme‐linked immunosorbent assay (ELISA) kits were acquired from CUSABIO (Wuhan, China). The TRAP staining kit was obtained from Solarbio (Beijing, China).

Proteins were lysed from cell samples by RIPA lysis buffer (Cat# P0013B, Beyotime, Shanghai, China) and quantified by a BCA protein concentration assay kit (Cat# PC0020, Solarbio, Beijing, China). Thirty micrograms of protein was loaded, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes for antibody blotting. The primary antibodies were ATG7 (Cat# 67,341–1-Ig) and BECN1 (Cat# 11,306–1-AP), purchased from Proteintech (Wuhan, China). GAPDH (Cat# ab59164, Abcam, Cambridge, MA, USA) was used as a control. Primary antibody incubations were performed at a dilution of 1:2000 in PBS for 16 h at 4 ℃. The secondary antibodies were purchased from Yeasen Biotechnology (Shanghai, China) and included peroxidase-conjugated goat anti-rabbit (1:5000 dilution, Cat# 33101ES60) and anti-mouse IgG (1:5000 dilution, Cat# 33201ES60). Secondary antibody incubations were 1 h at room temperature.

GNIP1/RNF90 (Abcam; ab170538), β-Actin (Proteintech; 66,009–1-lg), GAPDH (OriGene; TA802519), DDDDK Tag (Proteintech; 20,543-1-AP), LC3B (Proteintech; 18,725–1-AP), SQSTM1/p62 (Proteintech; TA502127), BECN1 (Proteintech; 11,306–1-AP), ULK1 (Cell Signaling Technology; 6439), HA-tag (Thermo Fisher Scientific; 26,183), GFP (Proteintech; 66,002-1-lg), UB antibody (Proteintech; 10,201–2-AP), K48 (Cell Signaling Technology; 8081), K63 (Cell Signaling Technology; 5621), PIK3C3/VPS34 (Thermo Fisher Scientific; PA5-35,215), 14–3-3ζ (Proteintech; 14,881-1-AP), anti-His (Proteintech; 10,001-0-AP/ 66,005-1-LG), anti-V5 (Proteintech; 14,440-1-AP). Cycloheximide (Sigma; C7698), DMSO (Sigma; D2650), MG132 (Biovision; 1791-5). Myc-DDDK-Tag-GNIP1, pcDNA3.1-Flag-GNIP1-W57A, PCDH-Flag-GNIP1, pEGFP-C2-LC3B, pCMV-HA-BECN1, pCMV-HA, pcDNA3.1-Flag-PIK3C3, pCMV-Flag-14-3-3ζ, pcDNA3.1-V5-BBCC, pcDNA3.1-V5-His-RBB and pcDNA3.1-V5-His-CCB plasmids were constructed by ourselves.

The protein was extracted with lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na₃VO₄, and 1 mM leupeptin cocktail]. Thirty micrograms of protein were loaded onto 10% polyacrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was transferred onto a nitrocellulose membrane, which was blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C.
After washing with TBST, the membrane was incubated with a secondary antibody for 1 h at room temperature. The signalling was detected with an enhanced chemiluminescence HRP substrate by Western blotting (Pierce, Rockford, IL, U.S.A.). The following primary and second antibodies were purchased from Proteintech (Wuhan, CHINA): PD-L1 (#28076-1-AP, 1 to 1000 dilution), S6K (#14485-1-AP, 1 to 2000 dilution), BECN1 (#11306-1-AP, 1 to 2000 dilution), p-AKT (# 66444-1-Ig, 1 to 1000 dilution), AKT (#10176-2-AP, 1 to 1000 dilution), LC3 (#18725-1-AP, 1 to 2000 dilution), GAPDH (# 10494-1-AP, 1 to 5000 dilution), HRP-linked anti-rabbit secondary antibody (#10256-2-AP, 1 to 5000 dilution) and HRP-linked anti-mouse secondary antibody (#10358, 1 to 5000 dilution). P-S6K (# 9204S, 1 to 1000 dilution) were purchased from Cell Signaling Technology, Inc (CST, U.S.A.).

This procedure was completed using the following primary antibodies raised against: FBXO22, GAPDH, P62, BECN1 and ATG5 (Proteintech Group); MMP-2, ERK, p-ERK, P38, p-P38, p-90RSK, RAC1 and BCL-2 (Cell Signaling Technology), and LC3 I/II (Abcam). The secondary antibodies were goat anti-rabbit and goat anti-mouse corresponding HRP (Beijing Biodragon Immunotechnologies Co., Ltd., Beijing, China). The protein bands were determined using a Tanon 5200 automated chemiluminescent imaging analysis system with ECL reagents (Tanon, Shanghai, China).

GBM tumour and mouse paraffin sections were dewaxed, rehydrated and antigen retrieval was performed. Sections were blocked with 3% hydrogen peroxide for 10 min and with normal goat serum for 30 min at room temperature. Then, the sections were incubated with anti-LRRC4 (Abcam), DEPTOR (Cell Signaling Technology), BECN1 (Proteintech) and LC3B (Cell Signaling Technology) antibodies for 12 h at 4 °C and were incubated with biotinylated secondary antibody (Maxim Biotechnologies) for 30 min at room temperature and streptavidin conjugated HRP for 30 min. Staining was visualized with 3,3-diaminobenzidine (DAB; Maxim Biotechnologies) and was counterstained with haematoxylin.