Trypan blue

Before infection, bMECs in a well were stained with 0.4% trypan blue (Beyotime Biotechnology, Beijing, China) and enumerated with a cell counter. The K. pneumoniae cultures were centrifuged for 15 min at 6000 g, washed once in sterile Phosphate Buffered Saline (PBS, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at pH 7.2, and re-suspended in DMEM, with concentrations adjusted to achieve multiplicity of infection (MOI) = 5. The MOI was defined as the ratio of added K. pneumoniae to bMECs.

Trypan blue (Beyotime, Shanghai, China) reflects the rupture of the cell membrane. The number of blue cells in each well was determined by a microscope and the percentage of positive cells was calculated.

The human HCC line HepG2 and CC cell line QBC939 were obtained from the cell bank of the Institute of Biochemistry and Cell Biology (Shanghai, China) and the Third Military Medical University, respectively. Both cell types were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA), supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) in a 5% CO₂ humidified environment at 37°C. RIPA lysis buffer, Trypan Blue and JC-1 was from Beyotime (Shanghai, China). Antibodies used were rabbit polyclonal p62 and anti-LC3 (1:1,000) from Abcam, anti-G6PDH (1:200) from Santa Cruz Biotechnology (Santa Cruz, CA), and mouse monoclonal anti-β-actin (1:1,000) and horseradish peroxidase-conjugated secondary antibodies (1:1,000) from Proteintech (Chicago, IL). MitoSOX Red mitochondrial superoxide indicator was purchased from Invitrogen (Carlsbad, CA). 2’,7’-Dichlorofluorescin diacetate (DCFH-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3-Methyladenine (3-MA), (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (Mito-TEMPO), chloroquine diphosphate salt (CQ), cisplatin, and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) were from Sigma-Aldrich (St. Louis, MO). 2-DG and DHEA was purchased from Aladdin industrial Corporation (Shanghai, China).

SAHA (Purity ≥99%) was purchased from Selleck Chemicals (Houston, TX). Matrigel and the anti-Semaphorin-4D (Sema-4D) antibody were obtained from BD Biosciences (San Jose, CA). Trypan blue was purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC apoptosis detection kit was purchased from Biotech Co., Ltd (Nanjing, China). RNase-free DNase I was from Qiagen (Hilder, Germany). RevertAid™ First Strand cDNA Synthesis Kit was purchased from Fermentas Life Sciences (Chicago, IL). Taq™ DNA Polymerase was from TaKaRa Biotechnology Co., Ltd (Dalian, China). Propidium iodide (PI), monoclonal antibody against β-actin and gelatin were obtained from Sigma (St. Louis, Mo). The anti-cyclin-D1 antibody was obtained from ABGENT (Suzhou, China). Anti-epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA). Akt, p-Akt (Ser 473), p70S6 kinase (S6K1), p-S6K1 (Thr 389), S6, p-S6 (Ser 235/236), mTOR, p-mTOR (Thr 289), Ulk1, p-Gsk-3β, Ulk1, Erk1/2 and p-Erk1/2 antibodies were purchased from Cell Signaling Tech (Beverly, MA). Primers were synthesized by GENEWIZ, Inc. (Suzhou, China).

The human HCC cell line HCCLM3 and mouse HCC cell line H22 were obtained from the China Center for Type Culture Collection, the mouse HCC cell line Hepa1–6 was obtained from Cell Bank of Type Culture Collection Chinese Academy of Sciences, authenticated by short tandem repeat (STR) analysis, and tested for mycoplasma contamination. HCCLM3 (abbreviated LM3) and Hepa1–6 cells were cultured in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (DMEM) and H22 cells were cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) supplemented with 10% foetal bovine serum, 2 mM l-glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin (all from Thermo Fisher Scientific, Gibco, Grand Island, NY, USA). All cells were maintained in a humidified incubator with an atmosphere containing 5% CO₂ at 37 °C.
The following reagents were used: sodium dichloroacetate (#347795; Sigma-Aldrich, St. Louis, MO, USA), trypan blue (#ST798; Beyotime Biotechnology, Shanghai, China), and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (#M2128; Sigma-Aldrich).