Fig 2: The influence of Rb1 on the Gas6/Axl signalling pathway. (A) Western blotting was performed to examine the protein expression of Gas6 and Axl. (B) qPCR was performed to examine the mRNA expression of Gas6 and Axl. The data are expressed as the mean ± SD. *p < 0.05, **p < 0.01 vs. the Young group; #p < 0.05 vs. the Old + Vehicle group.

Fig 3: Tumour intrinsic PD-L1 promotes Gas6 transcription and secretion.a, b qRT-PCR analysis of Gas6 and PROS1 mRNA expression levels in stable PD-L1 knockdown and overexpression cell lines. c The basal secretion level of Gas6 in PD-L1 stable cell lines and control cells. Data were presented as the mean ± SD. Data were analysed using non-paired Student’s t test, one-way ANOVA analysis followed by Bonferroni’s post hoc test. **P < 0.01; ***P < 0.001 vs. control or as indicated.

Fig 4: Tumour intrinsic PD-L1 regulates NSCLC cell proliferation through Gas6/MerTK signaling.a, b Western blot assay of PD-L1, p-MerTK, MerTK, p-AKT, AKT, p-Erk, Erk, Bcl-2 and Cyclin D1 expression in stable PD-L1 knockdown or overexpressed cells when compared to control cells. c, d PD-L1-overexpressed and control cells were treated with 3 µM UNC2025 or 10 µM LDC1267 for 48 h, then the cell lysates were proceed with western blot assay to test PD-L1, p-MerTK, MerTK, p-AKT, AKT, p-Erk, Erk, Bcl-2 and Cyclin D1 protein levels. e Western blot assay of the above proteins in PD-L1 overexpressing cells after knocked down with two separate si-MerTKs. f Western blot assay of p-MerTK, MerTK, p-AKT, AKT, p-Erk, Erk, Bcl-2 and Cyclin D1 protein levels in PD-L1 stable knockdown cells after stimulated with 500 ng/ml rh-Gas6 for 24 h.

Fig 5: PD-L1 cooperates with SP1 to regulate Gas6 transcription.a, b The results of the luciferase reporter assay in H1299-PD-L1-overexpressed cells and control cells after transfection with a set of luciferase reporter plasmids containing different regions of the Gas6 promoter or mutants at three SP1 binding sites for 24 h. c ChIP assays were performed to detect PD-L1 and Sp1 binding to the Gas6 promoter in H1299-PD-L1-overexpressed cells and control cells. d, e The mRNA and protein levels of Gas6 in H1299 and HCC827 cells after transient transfection with Sp1 siRNAs or Sp1-overexpressing plasmids. f The luciferase activity of the Gas6 promoter in 293T cells transfected with control vector, PD-L1 vector, Sp1 vector or both PD-L1 and Sp1 vectors. g H1299- and HCC827-PD-L1-overexpressed cells were silenced with si-Sp1 or si-NC (control). Data were presented as the mean ± SD. Data were analysed using non-paired Student’s t test, one-way or two-way ANOVA analysis followed by Bonferroni’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 vs. control or as indicated.