Fig 1: AXL inhibits sorafenib-induced immunogenic cell death, which could be rescued by TNF-α and IFN-α in sorafenib-resistant HCC.A WB analysis of secretory HMGB1 in the conditioned media of Sen and SoraRes HepG2 cells upon sorafenib treatment at the indicated concentrations. B ATP concentrations in the conditioned media collected from Sen and SoraRes HepG2 cells upon sorafenib treatment. C Representative FACS plots (left) and percentages (right chart) of membrane calreticulin expression in Sen and SoraRes HepG2 cells upon sorafenib treatment. D WB analysis of secretory HMGB1 in the conditioned media of Sen and SoraRes HepG2 cells treated with BGB324 at the indicated concentrations. E ATP concentrations in the conditioned media collected from Sen and SoraRes HepG2 cells treated with BGB324. F Representative FACS plots (left) and percentages (right chart) of membrane calreticulin expression in Sen and SoraRes HepG2 cells treated with BGB324. G WB analysis of secretory HMGB1 in the conditioned media of SoraRes HepG2 cells treated with either IFN-α or TNF-α alone or combined IFN-α and TNF-α in the presence or absence of sorafenib. H ATP concentrations in the conditioned media collected from SoraRes HepG2 cells treated with either IFN-α or TNF-α alone or combined IFN-α and TNF-α in the presence or absence of sorafenib. I Representative FACS plots (top) and percentages (bottom chart) of membrane calreticulin expression in SoraRes HepG2 cells treated with either IFN-α or TNF-α alone or combined IFN-α and TNF-α in the presence or absence of sorafenib. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant on a two-tailed unpaired Student’s t test for (B–F) and one-way ANOVA with Bonferroni’s multiple comparisons test for H, I.
Fig 2: Knockdown of PDPK1 phenocopies AXL inhibition in sorafenib-resistant HCC.A WB analysis of p-PDPK1, nuclear p-p65, nuclear histone H3, and TNF-α in SoraRes HepG2 cells with PDPK1 knockdown upon sorafenib treatment. B ELISA quantification of secretory TNF-α in SoraRes HepG2 cells with PDPK1 knockdown upon sorafenib treatment. C Representative FACS plots (left) and percentages (right chart) of mitoSOX staining in SoraRes HepG2 cells with non-target control (shNTC) or PDPK1 knockdown (shPDPK1-C2 and shPDPK1-C5). D qRT-PCR quantification of mt16S, mtDloop, and mtCYTB levels in the cytosol extract of SoraRes HepG2 cells with PDPK1 knockdown upon sorafenib treatment. E WB analysis of STING pathway in SoraRes HepG2 cells with PDPK1 knockdown. F WB analysis of IFN-α in SoraRes HepG2 cells with PDPK1 knockdown upon sorafenib treatment. G ELISA quantification of secretory IFN-α in SoraRes HepG2 cells with PDPK1 knockdown upon sorafenib treatment. H qRT-PCR quantification of mt16S, mtDloop, and mtCYTB levels in the cytosol extract of SoraRes HepG2 cells with PDPK1 knockdown. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s. not significant on one-way ANOVA with Bonferroni’s multiple comparisons test.
Fig 3: AXL inhibition induces TNF-α expression and promotes STING-type I interferon pathway.A Gene Set Enrichment Analysis (GSEA) of transcriptomes of AXL-low versus AXL-high HCC patients from TCGA-LIHC; left - Biocarta and right - Hallmark. B ELISA quantification of secretory TNF-α in sorafenib-sensitive (Sen) and sorafenib-resistant (SoraRes) HepG2 cells upon sorafenib treatment. C ELISA quantification of secretory TNF-α in Sen and SoraRes HepG2 cells upon BGB treatment. D Representative FACS plots (top) and percentages (bottom-right chart) of mitoSOX staining in Sen and SoraRes HepG2 cells upon BGB324 treatment. E qRT-PCR quantification of mt16S, mtDloop, and mtCYTB levels in the cytosol extract of Sen and SoraRes HepG2 cells upon BGB324 treatment. F WB analysis of STING pathway in SoraRes HepG2 cells upon BGB324 treatment. G ELISA quantification of secretory IFN-α in Sen and SoraRes HepG2 cells treated with BGB324. H qRT-PCR quantification of ISGs in Sen and SoraRes HepG2 cells treated with BGB324. I GSEA of AXL-high versus AXL-low HCC patients from TCGA-LIHC showing a negative correlation with interferon-stimulated gene signature. *p < 0.05; **p < 0.01; ****p < 0.0001; n.s. not significant on a two-tailed unpaired Student’s t test or one-way ANOVA with Bonferroni’s multiple comparisons test.
Supplier Page from Thermo Fisher Scientific for Recombinant Human IFN-alpha A/D Protein