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. 2019 Jan 15;10(5):573–583. doi: 10.18632/oncotarget.26542

Figure 4. Regulation of PD-L1 protein expression.

Figure 4

(A) Western blot and semiquanititative analysis of GSK-3beta and its inactivated state represented as phosphorylated-GSK-3beta (P-GSK-3beta) at Ser09 24h and 96h after irradiation with 8Gy. Non-IRR cells (0Gy) were used as controls. RR HNSCC cell lines did not show any significant change in GSK-3beta expression (lower left panel). However, inactivation of GSK-3beta occured 96h after irradiation. Phosphorylation at Ser09 was on average 10x higher compared to the level of non-IRR cells. Beta-Actin was used as loading control. n=3, Student’s t-Test ** = p < 0,01, ns=not significant (B) PD-L1 expression is dependent on GSK-3beta activation. Western blot analysis of PD-L1 expression after inhibition of GSK-3beta with 20μM LiCl and specific inhibition with 1μM and 5μM BIO. Exemplified GSK-3b dependent PD-L1 expression with HNSCC cell line PCI52. n=3. Ponceau staining was used as loading control. (C) Co-immunoprecipitation for identification of an interaction between PD-L1 and GSK-3beta without (0Gy) and after 96h of irradiation with 4 and 8Gy. GSK-3beta, including its attached binding partners, was precipitated with a specific antibody. Presence of PD-L1, necessarily interacting with GSK-3beta, was proven via western blot analysis. All RR cell lines PCI8, 15, 52 showed a decrease of PD-L1 interaction after irradiation. GSK-3beta detection served as loading control. Either a polyclonal rabbit IgG Isotype antibody, immunoprecipitation without antibody or without lysate was used as negative control. Undetectable GSK-3beta indicates no unspecific binding. (D) Semiquantitative analysis of western blot indicating relative interaction of GSK-3beta with PD-L1 in percent. After irradiation with 8Gy all RR cell lines PCI8, 15, 52 showed less interaction between GSK-3beta and PD-L1. PCI15 and PCI 52 also showed a significantly reduced interaction of GSK-3beta with PD-L1 after irradiation with 4Gy. Samples were normalized with GSK-3beta. Non-IRR 0Gy value of PCI8 was used for baseline definition. n=3, 2-way ANOVA * = p < 0,05, ** p= < 0,01.