Fig 1: IRF9 is an identified protein that interacts with apatinib and potentially plays a role in the development of drug resistance to apatinib in HNSCC(A) Schematic diagram of SPIA proteomics based on DIA quantification.(B) The SPIA results showed that apatinib binds to VEGFR2 and IRF9 in drug-resistant cells.(C) Heatmap displaying the binding levels of apatinib to proteins at different time points (top 15 p values).(D) IRF9 exhibits the highest binding affinity to apatinib during the development of apatinib resistance in HNSCC.(E) Molecular docking shows that apatinib has a strong binding ability with IRF9.(F) During the development of apatinib resistance in HNSCC, the binding affinity of VEGFR2 for apatinib remained largely unchanged.(G) Transcriptome sequencing analysis revealed the mRNA expression levels of VEGFR2 and IRF9 in different process of cells with apatinib. (H and I) The transcription and protein levels of VEGFR2 during different process of cell resistance by PCR and WB. (J and K) The transcription and protein levels of IRF9 during different process of cell resistance by PCR and WB. Data are represented as mean ± SEM. ns, not significant.
Fig 2: IRF9 affects JAK-STAT pathway(A) Heatmap for differential gene expression in JAK-STAT pathway.(B and C) qRT-PCR and western blot validated the expression of SOCS2, PIM1, CCND1/2, and MYC in sh_IRF9 and control group.(D) Interaction of IRF9 with STAT1 and STAT2.(E and F) IF microscopy analysis of the effect of sh_IRF9 induced STAT1 and STAT2 relocalization. Scale bars, 20 μm.(G) Example integrative genomics viewer (IGV) tracks of IRF9 ChIP-Seq, input controls, and RNA-Seq of IL10 and JAK3.(H and I) qRT-PCR and western blot validated the expression of IL10 and JAK3 in sh_IRF9 and control.(J) Images and quantification of western blot for p-STAT3 Y705, p-STAT3 S727, and STAT3 after inhibition of IRF9 in HNSCC. Data are represented as mean ± SEM. ns, not significant, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 3: IRF9 promotes tumor cell proliferation, survival, and invasion in HNSCC(A)Western blot validated the expression of IRF9 in sh_IRF9 and ov_IRF9.(B) Migration and invasion ability of HNSCC cell lines in sh_IRF9 and ov_IRF9. Scale bars, 200 μm.(C) Colony formation assay.(D) EdU assay determined the EdU positive rate. Scale bars, 200 μm.(E) Apoptosis assay.(F) Cell-cycle assay.(G) The tumor size in nude mice subcutaneously treated with sh_IRF9 and ov_IRF9 HNSCC cells.(H) Size of lung in the lung metastasis model.(I) Ki67 and VEGFR2 expression in lung metastasis tumors after IRF9 knockdown, then increased after IRF9 overexpression. HSC-3R: the HSC apatinib-resistant cell line, CNE-2R: the CNE-2 apatinib-resistant cell line. Data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 4: Oroxin B inhibits tumor progression in HNSCC(A)The results of molecular docking simulations of IRF9 with compound five small molecule drugs.(B) Western blot validated the expression of STAT3 and p-STAT3 for five small molecule drugs.(C) Kinetic analyses of the binding between IRF9 and the tested drugs (HY-N1435 and Z56772943) measured by an SPR-based Biacore instrument. The derived dissociation constants between IRF9 and the tested drugs (HY-N1435 and Z56772943) are also shown.(D and E) Migration and invasion ability of apatinib-resistant HNSCC cell lines treated with Oroxin B (HY-N1435).(F) Colony formation assay.(G) EdU assay determined the EdU positive rate. Scale bars, 100 μm.(H) The tumor size in nude mice subcutaneously treated with Oroxin B.(I) In vivo fluorescence imaging of cancer cell tail vein injection into mice.(J) Size of lung in saline and Oroxin B injected into tumor-bearing mice.(K) Ki67, MYC, TCF7, and p-STAT immunohistochemical staining of lung metastasis. Scale bars, 100 μm. HSC-3R: the HSC apatinib-resistant cell line, CNE-2R: the CNE-2 apatinib-resistant cell line. Data are represented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
Fig 5: High IRF9 indicated poor overall survival(A) High IRF9 and low IRF9 were identified and immunohistochemical staining. Scale bars, 100 μm.(B) PFS of low/high IRF9 HNSCC patients treated with apatinib-based combination therapies.(C) The difference in the number of low/high PFS in HNSCC patients treated with apatinib-based combination therapies with low/high IRF9.(D) The difference in the number of low/high PFS in HNSCC patients treated with apatinib-based combination therapies with low/high nuclear-to-cytoplasmic ratio of IRF9.(E) The pathogenesis diagram of the development of apatinib resistance in HNSCC.
Supplier Page from OriGene Technologies for Interferon regulatory factor 9 (IRF9) Human shRNA Lentiviral Particle (Locus ID 10379)