Fig. 2.

C-FOXP3 directly activates PD-L1 transcription in PDAC cells. a Establishment and verification of stable cell lines overexpressing or underexpressing FOXP3. pLV-control and pLV-FOXP3 indicate lentivirus vectors for the control and overexpression of C-FOXP3; pLKO-control and pLKO-FOXP3 indicate lentivirus vectors for the control and knockdown of C-FOXP3. (Left) Representative samples of C-FOXP3 and PD-L1 in Panc-1 and Pan02 cell lines detected by western blotting. GAPDH was used as an internal control. (Right) The protein and mRNA levels of PD-L1 were measured by western blot (GAPDH as a control) and real-time PCR (β-actin as a control). Histogram (columns: mean, bars: standard deviation, n = 3), p values were calculated by Student’s t-test, *p < 0.05, **p < 0.01. b Pancreatic cancer cell lines were stained with IgG isotype control and PD-L1-specific monoclonal antibody and analyzed for PD-L1 protein levels by flow cytometry. Quantification of PD-L1 protein levels on different pancreatic cancer cell surfaces. Columns: mean, bars: standard deviation, n = 3, p values were calculated by Student’s t-test, *p < 0.05, **p < 0.01. c (Upper) Specificity of the ChIP assay. The human PD-L1 gene, including the FOXP3 binding motif a to c. (Lower) Binding of FOXP3 and the PD-L1 promoter was observed at motifs a and b by PCR analysis. P21 was used as a positive control. N: negative control; P: positive control. d Panc-1 cells were transfected with either vector control or FOXP3 in conjunction with the luciferase reporter pGL3-PD-L1 (wild type), pGL3-motif a MUT or pGL3-motif b MUT (mutation of motif-a or motif-b) vectors. The pGL3-p21 promoter was used as a positive control. After 48 h, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter assay (Promega), and the ratio was determined. Histogram (columns: mean, bars: standard deviation, n = 3). p values were calculated by Student’s t-test, *p < 0.05