Figure 4.

PD-L1 expression in adipose tissue is regulated by IFNγ but is not increased in the setting of cancer. (A) Immunoblot depicting the expression of brown fat marker uncoupling protein 1 (UCP1), PD-L1, and beta actin in the whole tissue lysate (‘whole’) and stromovascular fraction (‘SVF’) of brown adipose tissue (BAT) and visceral adipose tissue (VAT). Samples were taken from Pdl1+/- (‘WT’) and Pdl1-/- (‘KO’) mice. The band corresponding to PD-L1 is indicated with arrows. (B-C) Western blot depicting the expression of PD-L1 and beta actin in VAT (B) and BAT (C) isolated 24 hours after the systemic administration of PBS, 1B7, B3-IFNγ, or 1B7-IFNγ. (D-E) RT-qPCR analysis depicting the expression of Irf1 and Pdl1 in the VAT (D) and BAT (E) isolated 24 hours after the systemic administration of PBS, 1B7, B3-IFNγ, or 1B7-IFNγ. The normalized expression ratio (NER) relative to the housekeeping gene beta-Actin is shown. (F) Comparison of Pdl1 expression by RNA-seq in VAT of sham-operated and KPC tumor-bearing mice. Values plotted are DESeq2-derived normalized expression values. The difference in Pdl1 expression between these groups was not statistically significant (P = 0.71). (G–H) Western blot depicting the expression of PD-L1 and beta actin in VAT (G) and BAT (H) of sham-operated and KPC tumor-bearing mice. Statistical analyses in (D) and (E) were performed using an ordinary two-way ANOVA with Dunnett’s multiple comparisons test; mean ± SD is shown. Immunoblot in (A) was blocked with 3% BSA in PBST, while subsequent PD-L1 blots were blocked with 5% BSA in PBST.