Aim 5 albumax

MNT1 cells were from Prof. Tiechi Lei (Renmin Hospital of Wuhan University) and grown in MEM medium (SH30024.01; HyClone) supplemented with 20% fetal bovine serum (10099-141; GIBCO), 10% AIM-V+AlbuMAX (31035025; GIBCO), 1% sodium pyruvate (11360070; GIBCO), and 1% Non-Essential Amino Acids Solution (100×) at 37°C and 5% CO₂. Melan-a cells were from the laboratory of Dorothy C. Bennett (St. George’s University) (Bennett et al, 1987 (link)) and grown in DMEM medium (11965092; GIBCO) supplemented with 10% fetal bovine serum (10099-141; GIBCO) at 37°C and 10% CO₂. siRNAs (Table S1) were transfected into cells to knock down VDAC1 using Lipofectamin RNAiMAX Transfection Reagent (13778100; Invitrogen) according to the manufacturer’s instructions.

Table S1 siRNA sequences used in knockdown experiments.

Human peripheral blood mononuclear cells were isolated and purified from peripheral blood of healthy human donors using Lymphoprep (Stemcell, Canada). Cells were then activated with 100 ng/mL human anti-CD3 (clone OKT3; Sino Biological, Beijing, China) and 100 U/mL IL-2 (Sino Biological) in AIM-V^® + AlbuMAX^® (bovine serum albumin) Serum-free Medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) for 48 hours at 37°C in a 5% CO₂ incubator. T cells were expanded for 10-20 days at a concentration of 1×10⁶ cells/mL in the presence of continuous IL-2 stimulation.
After activation for 48 hours, T cells were transduced with CAR retroviral vector and cultured in a 5% CO₂ incubator at 37°C for 48 hours. The transduction efficiency was detected by flow cytometry analysis (CytoFLEX, Beckman Coulter, USA). The retroviral vector copy number integration per T cell was detected by qPCR; the primer sequences were provided in Supplementary Table 1.