Trypsin

MEFs were isolated from pregnant C57BL/6 J mice at 13.5 - day post coitum. The extraction process was in accordance with a previously described protocol⁵⁶ : Embryos were isolated from embryo sacs, and heads and limbs were removed. The remaining tissues were subjected to trypsinization in 0.05% trypsin/EDTA (Gibco, 25300-062) with DNAase I (Solarbio, D8071). trypsin was inactivated by adding Dulbecco’s modified Eagle’s medium (DMEM, Gibco, C11995500BT), and cells were collected through centrifuging at low-speed (799 g, 5 min). The isolated MEFs were cultured on plates coated with 0.1% gelatine (sigma, G9391-100G) for 12 h and frozen when reaching approximately 90% confluency (P0). MEFs were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco, 10099-141) and 1% penicillin/streptomycin (Yeasen, 60162ES76), and passaged when reaching 90% confluency. Replicative senescent MEFs (P7) were used for further assays.
MRC-5 cells were obtained from Cell Bank of Chinese Academy. MRC-5 cells were grown in Minimum Essential Medium (MEM, Gibco, 11090-081) with 10% FBS (Biochannel, BC-SE-FBS01), 1% penicillin/streptomycin (Yeasen, 60162ES76), 1% sodium pyruvate solution (Gibco, 11306-070), 1% GlutaMAX (Gibco, 335050-061) and 1% nonessential amino acids solution (Gibco, 11140-050). MRC-5 cells were treated with Dox (100 nM) every other day for the first three days, and cultured for the next four days to induce senescence.^{57 (link)} Drugs to be tested were added to MEM during the process. For replicative senescence, MRC-5 cells passaged to more than P42 was used.
NRK-52E cells were obtained from Cell Bank of Chinese Academy. NRK-52E cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco, 10099-141) and 1% penicillin/streptomycin (Yeasen, 60162ES76), and passaged when reaching 90% confluency. NRK-52E cells were treated with MMC (0.5 μM) for 2 days, and cultured for the next 2 days to obtain MMC-induced senescence.^{37 (link)} Drugs to be tested were added to DMEM after removal of MMC, and kept for 2 days before assays.
H1299 cells HEK293T cells were cultured in RPMI 1640 medium (Corning, 10-040-CV) supplemented with 10% fetal bovine serum (Vivacell, C04001-500). To generate stable cell lines for subcellular lactate detection, the pLVX lentiviral plasmids encoding FiLa sensors were constructed. Lentivirus was produced by co-transfecting two lentiviral packaging vectors (pMD2.G and psPAX2) in HEK293T cells. Lentiviral supernatants were collected 48 and 72 h after transfection. H1299 cells in 6-well tissue culture plates were infected in media containing 8 mg/mL polybrene. After infection, the virus was removed, and cells were selected with 0.2-1 µg/mL puromycin for one week to obtain stable cell lines.

Overnight bacterial cultures were washed with Dulbecco’s Phosphate Buffered Saline (DPBS, Sigma, D8537) and resuspended in Bifidobacterial medium. HRT18 cells were seeded in a 6-well plate and grown until full confluency. The cells were then transferred to the anaerobic chamber, washed twice with anaerobic DPBS to remove remaining oxygen, and infected with bacteria at a MOI of 10 and 50. After 2 h incubation at 37 °C, cells were washed 2 times with DPBS, incubated with 500 µL 0.25% trypsin (ThermoFisher, 25200-072) for 10 min at room temperature, and stopped with 500 µL of Dulbecco’s modified Eagle’s medium (DMEM) + glutamax (Life Technologies, 31966047) containing 10% fetal calf serum (FCS) (Sigma, F7524). The cell suspension was used to make tenfold serial dilutions. 100 μL aliquots from undiluted to the 10^–5 dilution were plated and colonies were grown at 37 °C for 1 or 2 days under anaerobic conditions. For all bacterial strains, visual colonies from dilutions 10^–3 or 10^–4 were counted to determine CFU/mL.

Primary HCC cells were isolated from tumor masses grown on genetically modified mice with the following procedures. The liver tumors were rinsed once with 1 × PBS and ground into pieces. Collagenase type I in PBS (2 µg/mL) was added and placed in a 37°C incubator (Sanyo, Osaka, Japan) for 2 h with constant shaking. The tissue solution was then incubated with 0.25% trypsin (Gibco, Gaithersburg, MD, USA) for 5 min at 37°C, followed by centrifugation at 300 rpm. Next, the supernatant was gently passed through a 100 µm sterile filter, and the filtrates containing tumor cells were centrifuged (Hitachi, Tokyo, Japan) at 1200 rpm for five minutes. Finally, cell pellets were cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin.

All chemicals and reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China) and were used without further purification unless stated specifically. Neurobasal medium, trypsin and B27 were purchased from Thermo Fisher Scientific (U.S.A.).