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. Author manuscript; available in PMC: 2025 Jun 3.
Published in final edited form as: Mol Cancer Ther. 2024 Dec 3;23(12):1801–1814. doi: 10.1158/1535-7163.MCT-24-0369

Figure 6. TBK1 inhibitor uncouples the cell cycle inhibitory response from the IFN response.

Figure 6.

A. ISRE-mCherry reporter assay representative images of HT1080 cells treated with DMSO, CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM), TBK1i (GSK8612, 5 μM) or in combination. Poly (I:C) was used as a positive control for dsRNA-activated IFN response as measured by the ISRE-mCherry reporter system. Scale bar = 400 μm. B. qPCR analysis of STAT2 and IRF9 IFN-related gene expression in HT1080 cells treated with vehicle, Poly (I:C) alone or in combination with TBK1i (GSK8612, 5 μM). C. qPCR analysis of STAT2 and IRF9 IFN-related gene expression in HT1080 cells treated with DMSO or CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM), with and without TBK1i (GSK8612, 5 μM). D. Immunoblot analysis for TBK1, pTBK1, and cell cycle proteins in HT1080 cells treated with DMSO, CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM), TBK1i (GSK8612, 5 μM) or in combination. E. qPCR analysis of STAT2 and IRF9 IFN-related gene expression in HT1080 cells treated with DMSO, CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM) with and without TBK1i (GSK8612, 5 μM). F. Bubble plot of gene ontology analysis for both CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM) and triple-treatment (Palbociclib + Trametinib + GSK8612) groups using GSEA and KEGG database (biological process) of the top 25 downregulated (blue) and upregulated (red) pathways. G. Venn diagram displaying distribution of significantly differentially expressed genes in 519 cells treated with CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM) or triple treatment with TBK1i (GSK8612, 5 μM) included. ENRICHR analysis identifying shared enriched pathways downregulated in 519 cells treated with CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM) and triple treatment with TBK1i (GSK8612, 5 μM) included or uniquely expressed genes in 519 cells treated with CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM). H. Heatmap of select cell cycle and IFN-related gene expression in HT1080 cells treated with CDK4/6i (Palbociclib, 100 nM) and MEKi (Trametinib, 25 nM) ± TBK1i (GSK8612, 5 μM). Data displayed as mean ± SD in triplicate. **** p < 0.0001 as determined by one-way (B) and two-way ANOVA (C, E).