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. 2017 Jul 20;6(11):e1353860. doi: 10.1080/2162402X.2017.1353860

Figure 2.

Figure 2.

GDNF incubation stimulates PD-L1 mRNA and protein expression. (A) Real-time PCR assays to demonstrate PD-L1 mRNA upregulation in HNSCC cells. Cell lines were treated with vehicle control, NT-3 (30 ng/mL), NGF (30 ng/mL), GDNF (30 ng/mL), ARTN (30 ng/mL), BDNF (30 ng/mL) or NRTN (30 ng/mL) for 24 hours. (B) The flow cytometry examination indicated that GDNF increased PD-L1 protein expression. Cell lines were treated with vehicle control, ARTN (30 ng/mL), GDNF (30 ng/mL) or NGF (30 ng/mL) for 48 hours. (C) Representative flow cytometry analysis of PD-L1 on HN6, HN4 and HN30 cells in (B). (D) Western blot detection showed that PD-L1 protein expression was increased under stimulation. Cell lines were treated with a vehicle control, GDNF (30 ng/mL) or IFN-γ (20 ng/mL) for 48 hours. (E) A real-time PCR assay indicated that RET inhibitors abrogated GDNF-induced PD-L1 upregulation. Cell lines were treated with vehicle control, RETi (5 μmol/L), GDNF (30 ng/mL), or their combination for 24 hours. ***, p < 0.001 compared with the vehicle group. (F) PD-L1 protein expression was determined by flow cytometry after cells were treated with vehicle control, RETi (5 μmol/L), GDNF (30 ng/mL), or their combination for 48 hours. ***, p < 0.001 compared with the control group.