Figure 2.

Thyroid adenoma associated gene (THADA) interacted with programmed death-ligand 1 (PD-L1) to maintain its Golgi residency. (A) Duolink assay showing the interaction of endogenous THADA and PD-L1 proteins in RKO cells. Red dots indicating the binding of two proteins. Nuclei were stained with DAPI. Scale bar, 10 µm. Values are means±SD from three random fields in each treatment. Statistical differences were evaluated by two-sided Student’s t-test. Co-immunoprecipitation (Co-IP) assay detecting the interaction between THADA and endogenous PD-L1 (B) and exogenous PD-L1 (C), respectively, in the indicated cells. The experiments were repeated three times independently with similar results. (D) ELISA showing the direct interaction between purified proteins of THADA-His and PD-L1-FC (1 µg/mL). Programmed cell death 1 (PD-1) was served as a positive control (1 µg/mL). Statistical differences were evaluated by analysis of variance (ANOVA) post hoc test (Tukey). ***P=0.0001; ****p<0.0001; ns, no significance. (E, F) Immunofluorescence assays showing the co-localization of PD-L1 and THADA in RKO cells with indicated treatment. Green, PD-L1; red, THADA; blue, DAPI nucleus staining. Scale bars, 10 µm. Values are means±SD from n=3 independent experiments. The p values were evaluated by two-sided Student’s t-test. Immunofluorescence assays showing the co-localization of PD-L1 and GRP94 (G)/58K (H) in RKO cells transfected with THADA small interfering RNAs (siRNAs). Scale bars, 10 µm. White dashed boxes denote the representative fields to be magnified. (I) Quantification of the co-localization between PD-L1 and endoplasmic reticulum (ER)(GRP94)/Golgi(58K) in RKO cells. Values are means±SD from n=3 independent experiments. The p values were evaluated by two-sided Student’s t-test. (J) Western blot analyses showing the effect of THADA depletion on PD-L1 proteins extracted from total cell, the ER and Golgi apparatus with MG132 treatment (10 µM, 6 hours).