Figure 4. The C-terminal portion of PD-L1 protects TβRI mRNA from degradation.

(A) Actinomycin D (5 μg/mL) was added in cell culture to block gene transcription and cells were collected for qRT-PCR for TβRI mRNA at different time points. Knockdown of PD-L1 by two different shRNAs consistently accelerated degradation of TβRI mRNA in HSCs. *p < 0.05, **p < 0.01 by ANOVA, n = 3.

(B) Upper: anti-PD-L1 was used for RNA immunoprecipitation (RIP) to pull down PD-L1 protein and co-precipitated RNA was quantitated by qRT-PCR. Non-immune IgG was used as the control. TβRI mRNA co-immunoprecipitated with PD-L1 protein. ***p < 0.001 by ANOVA, n = 3. Lower: fluorescence in situ hybridization for TβRI mRNA (red) and IF for FLAG-PD-L1 FL protein were performed on the same cells. TβRI mRNA and FLAG-PD-L1 FL protein co-localized in the cytoplasm of a HSC (yellow, arrows). Scale bar, 20 μm.

(C) FLAG-tagged PD-L1 domains were introduced into PD-L1 knockdown HSCs and cells were collected for RIP using anti-FLAG. TβRI mRNA co-precipitated with PD-L1 FL or PD-L1 C + T by RIP. ***p < 0.001 by ANOVA, n = 3.

(D and E) FLAG-tagged PD-L1 domains were introduced into PD-L1 knockdown HSCs by retroviral transduction and cells were collected for WB (D) and qRT-PCR (E) for TβRI. PD-L1 FL and PD-L1 C + T rescued TβRI mRNA and TβRI protein of PD-L1 knockdown HSCs. **p < 0.01, ***p < 0.001 by ANOVA, n = 3.

(F) FLAG-tagged PD-L1 domains were introduced into PD-L1 knockdown HSCs followed by stimulation without or with TGF-β1 (5 ng/mL) overnight (upper) or 30 min (lower). Cells were collected for WB for HSC myofibroblatic activation markers or P-SMAD3. PD-L1 FL, but not the PD-L1 Ex and PD-L1 T + C mutants, rescued the impaired TGF-β signaling and myofibroblastic activation of PD-L1 knockdown HSCs. *p < 0.05, **p < 0.01, ****p < 0.0001 by ANOVA, n = 3. All data are represented as mean ± SEM.