Fig. 5. PTPRD directly dephosphorylates STAT3 and promotes STAT3-dependent ATG5 transcription.

A GSEA of the “IL-6/JAK/STAT3 signaling” gene modules in NPC patients with high or low PTPRD expression. FDR q false discovery rate q value, NES normalized enrichment score. B Western blotting analysis of p-STAT3 and STAT3 protein levels in PTPRD-overexpressing or -knockdown cells. These data are representative of three independent experiments. C 293T cells treated with IL-6 and co-transfected with flag-tagged PTPRD were used for immunoprecipitation. PTPRD and p-STAT3 protein levels were detected via western blotting. These data are representative of three independent experiments. D Immunofluorescence staining of ectopically expressed PTPRD and STAT3 in HONE1 and HK1-EBV cells. E Expression of p-STAT3, STAT3, BECN1, and ATG5 were detected in NPC cells treated with stattic or IL-6. GAPDH was used as an internal control. F ChIP analysis for detection of p-STAT3 binding to the ATG5 promoter in HONE1 and CNE2 cells after treatment with Stattic (20 Μm) and IL-6 (40 ng/mL) respectively. These data are representative of three independent experiments. Mean ± SD (n = 3); Student’s t tests; ***P < 0.001. G Schematic illustration of the 2-kb region upstream of the transcription start site of ATG5. Two potential binding sites of STAT3 at −335 to −329 and 97–103 were identified and labeled as A and B, respectively. H 293T cells transfected with the wild-type or mutant pGL4-ATG5 promoter were treated with IL-6 and subjected to dual-luciferase reporter assays. These data are representative of three independent experiments. Mean ± SD (n = 3); Student’s t tests; **P < 0.01; ***P < 0.001. I HONE1 and HK1-EBV cells stably expressing PTPRD or the control vector were treated with IL-6 and exposed to radiation. The protein levels of PTPRD, p-STAT3, STAT3, ATG5, LC3I/II, and P62 were detected using western blotting. GAPDH was used as an internal control. These data are representative of three independent experiments.