Mitomycin c

AFMSCs were pre-treated with 10 µg/mL mitomycin C (Med Chem Express) for 3 h. PBMCs isolated from healthy donors via Ficoll-Paque (GE) were labeled with the fluorescent dye 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) (Invitrogen). AFMSCs and PBMCs were then co-cultured at a 1:5 ratio for 72 h in RPMI-1640 (CORNING) supplemented with 30% FBS (Gibco) and 100 μg/mL PHA (Dahui Biotechnology). PBMCs proliferation were evaluated by measuring CFSE fluorescence via flow cytometry, and mRNA expression was examined using RT-qPCR.

For proliferation assays, primary CD4⁺ T cells expressing the CARs or control CD4⁺ T cells were washed with PBS and then labeled with CellTrace Violet Kit (Life Technologies) at the final concentration of 5 μM, according to the manufacturer’s instructions. Cognate target cells (AsPC-1) expressing desired antigens and non-cognate target cells (HT29, U87, or PANC-1) were treated with 25 μg/mL mitomycin C (MedChem Express), resulting in target cells with a replication-incompetent state. Then, labeled T cells were co-cultured with treated target cells at a 2:1 effector/target ratio in human T cell medium and mixtured cells were collected for flow cytometry analysis. Lastly, the proliferation of CAR-T cells was assayed by monitoring the CellTrace Violet dilution after incubation for 3 days.

Female C57BL/6 mice bearing a subcutaneous MC38 tumor of 150–200 mm³ were injected once intravenously with vehicle (saline), blank LNP or JCXH-211m (10 μg). Spleens were collected 2 weeks after treatment. Single splenocyte suspension (2 × 10⁶) was co-cultured with mitomycin C-treated (MedChemExpress, NJ, USA) MC-38 or B16F10 tumor cells (2 × 10⁴) in presence of anti-CD28 and anti-CD49d antibodies (BD Pharmingen, NJ, USA) for 18 h. Golgi transporter inhibitor (Invitrogen) was added in the co-culture during the last 16 h of incubation. The percentage of CD8 + IFN-γ + and CD4 + IFN-γ + cells were determined by flow cytometry after performing intracellular cytokine staining. Data are presented as mean ± SD, each dot represents one individual animal (n = 6 per group).

Human umbilical vein cells (HUVECs, PromoCell, C-12208) were cultured in endothelial cell growth medium (PromoCell, C-22010). To knockdown the expression of PTBP1, HUVECs were infected with lentivirus-shRNA-PTBP1 (LV-sh-PTBP1) (Gene Pharma, Inc.) at a multiplicity of infection (MOI) of 50 at 37 °C for 24 h, and then cultured with fresh medium for another 48 h at 37 °C in a 5% CO2 incubator before subsequent experiments. The cells infected with control lentivirus-shRNA (Gene Pharma, Inc.; MOI, 50) were used as a negative control. For the migration assay, HUVECs were incubated with 10 μg/mL mitomycin C (MedChemExpress) at 37 °C in a 5% CO2 atmosphere for 2 h^{50 (link)}. The LV-sh-PTBP1 sequences were as follows: 5’-CGGCACAGTGTTGAAGATCAT-3’. For the overexpression of ARRB1-L or ARRB1-S, HUVECs were infected with ARRB1-L overexpression lentivirus or ARRB1-S overexpression lentivirus. At 72 h after infection, the growth area was scratched. The cells were then photographed in five different fields at 0 h and 20 h after scratching using a Leica DMi8 microscope, respectively. Cell migration ability was calculated as a percentage of the wound area difference between 0 h and 20 h to the wound area at 0 h by ImageJ software.

Two straight lines were drawn at the back of the culture plate using a marker. The transfected cells were cultured into the plate individually and incubated until the cell growth density reached 90% confluence, and then treated with mitomycin C (1 µg/mL, MedChemExpress, HY‐13316) for 1 h to inhibit the proliferative effect of cells. Another set of lines was drawn on the cells, perpendicular to those at the back of the plate, using 200‐µL tips. The unbound cells were washed off, and the cells that remained on the plate were transferred to a DMEM medium containing 2% FBS. Subsequently, the regions where the lines intersected were photographed under a microscope. After 48 h, these regions were again observed under a microscope and photographed.