Anti lc3b

Cells or tumor tissue extracts were digested in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL). The proteins were quantified using a BCA protein assay kit (Thermo Scientific) according to the manufacturer's instructions. The total protein extracts were separated by 10% SDS‐PAGE and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies, including anti‐LC3B (Abcam, Cambridge, UK), anti‐ERK1/2 (Cell Signaling Technology CST, Danvers, MA), anti‐p‐ERK1/2(CST), anti‐mTOR (CST), anti‐p‐mTOR (CST), anti‐p85 (Abcam), anti‐p‐p85 (Abcam), anti‐Akt (CST), anti‐p‐Akt (CST), anti‐p70S6K (CST), anti‐p‐p70S6K (CST), anti‐p62 (CST), anti‐Craf (Abcam), anti‐p‐Craf (Abcam), anti‐Atg7 (CST), anti‐GAPDH and β‐actin (Abmart, China) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Abcam) for 1 h at room temperature, and the protein bands were detected with a ChemiDoc^™ XRS+ and Image Lab TM software (Bio‐Rad, Hercules, CA).

RAW264.7 macrophages (1 × 10⁵ cells/well in 12-well plates) were seeded overnight and washed twice with PBS, treated for 2–4 hours, washed in PBS, and then scraped into cold PBS. Starvation was performed in HBSS. BafA1 (100 nm), 1-TbAd (10 μM), or chloroquine at 10 μM treatment was followed by lysis (100 μL radioimmunoprecipitation assay buffer, MilliporeSigma) with protease and phosphatase inhibitor cocktail, Thermo Fisher Scientific) on ice for 10 minutes. Nuclei were pelleted at 13,000g for 5 minutes, and the supernatant was decanted. NuPAGE reducing agent and sample buffer (Thermo Fisher Scientific) were added, and the samples were boiled at 95°C for 10 minutes. The samples were run on 4%–12% SDS-PAGE gels and transferred onto PVDF membranes using iBlot2 and then blocked in 5% milk in TBS with 0.05% Tween-20 (TBS-T) for 1 hour at room temperature. Primary antibodies in 5% BSA in TBS-T were incubated overnight at 4°C, and then the membranes were washed 3 times in TBS-T. HRP-conjugated secondary antibodies were incubated for 1 hour at room temperature, washed 3 times before developing with enhanced chemiluminescent reagent (Merck) and imaging (Amersham AI600). The following antibodies were used: anti-LC3B (Abcam, 1:1,000), anti-p62 (Cell Signaling Technology, 1:1,000), anti–actin HRP (Cell Signaling Technology, 1:5,000), and anti–rabbit HRP (Promega, W4011, 1:10,000).

The harvested cells were fixed in 4% paraformaldehyde for 10 min and permeabilized with 1% Triton X-100 in PBS. The cells were incubated in 10% bovine serum albumin (BSA) and anti-LC-3B (Abcam #ab48394, Cell Signaling Technology, Danvers, MA, USA) at a 1:400 dilution after serial washes with PBS. Cells were washed with PBS and incubated with the secondary antibody, rhodamine Red^TM-X-conjugated goat anti-rabbit IgG (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). The cells were then incubated with Hoechst 33342 (Sigma-Aldrich) to identify the cell nuclei.

The IHC protocol was performed as described previously [15 (link)]. Briefly, tumour sections of patients and nude mouse xenografts were analysed by IHC using the EnVision™ System (Dako, Carpinteria, CA). Primary antibodies used for IHC were anti-BDH2 (1:100, Proteintech, 27,207), anti-Ki67 (1:100, Abcam, ab15580), anti-cleaved caspase-3 (1:200, CST, #9661), anti-LC3B (1:200, Abcam, ab48394), anti-Nrf2 (1:50, Proteintech, 16,396), anti-p-Akt (1:100, Proteintech, 66,444), and anti-p-mTOR (1:100, Abcam, ab109268).

Intracellular p62/SQSTM1 and LC3B staining was performed using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience) to permeabilize the cells. After being washed, the cells were incubated with anti-p62/SQSTM1 (Abcam) and anti-LC3B (Abcam) antibodies on ice for 15 min, followed by incubation with secondary antibodies on ice for 15 min.

RIPA buffer (Beyotime) containing phosphatase and protease inhibitors was used to lyse cells for 5 min. The resultant lysates were spun for 15 min at 12,000 g, and a BCA Assay Kit (Beyotime) was used to quantify protein levels. Samples were then boiled for 15 min and separated via SDS-PAGE before transfer onto PVDF membranes (Thermo Fisher Scientific). Blots were then blocked using 5% non-fat milk in TBST, probed overnight with anti-p-PI3K (Cell Signaling Technology, Boston, MA, USA), anti-LC3B, anti-p62, anti-PI3K, anti-Akt, anti-p-Akt (Abcam, Cambridge, UK) and anti-β-actin (Cwbio, China) at 4°C, washed thrice in TBST, probed with secondary HRP-linked antibodies for 1 h, and assessed via chemiluminescent analysis (Bio-Rad). Samples were analyzed in triplicate.

Cells were harvested following treatment and cellular proteins isolated using standard procedures. Proteins were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). Membranes were blocked with 5% skim milk for 60 min and then incubated with the following primary antibodies overnight at 4°C: anti-HIF-1α (1:1000, ABclonal, China, A11945), anti-LC3B (1:1000, Abcam, United Kingdom, ab48394), anti-BNIP3 (1:500, ABclonal, China, A5683), anti-FUNDC1 (1:500, ABclonal, China, A16318), anti-P62 (1:10000, Abcam, United Kingdom, ab109012), and anti-β-actin (1:10000, Bioss, China, bs-0061R). Blotted membranes were then incubated with 1:10000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (bs-0295G-HRP), and target bands visualized using an enhanced chemiluminescence detection system. Band intensities were calculated from gray scale images using ImageJ (Version 1.51). Target protein band intensities were normalized to β-actin band intensity (the gel-loading control). The ratio of cytosolic inactive LC3 (LC3-I) to lipidated LC3 (LC3-II) was calculated as an index of autophagic activation.