Figure 8.

KDM5C interacts with TBK1 to inhibit its phosphorylation and the expression of type I IFNs and ISGs. A) The number of peptide spectrum matches (PSMs) and unique peptides for TBK1 protein identified to be associated with KDM5C in the cytosol as described in Figure 5A was shown. B) Cytosolic extracts from MCF7 cells were subjected to immunoprecipitation (IP) with anti‐TBK1 antibody followed by immunoblotting (IB) analysis as indicated. C) Cytosolic extracts from HEK293T cells transfected with Flag‐tagged TBK1 and HA‐tagged KDM5C or ZMYND8 were subjected to immunoprecipitation (IP) with anti‐HA antibody followed by immunoblotting (IB) analysis as indicated. D) In vitro pull‐down assay was performed by mixing in vitro purified Flag‐tagged TBK1 and HA‐tagged KDM5C or ZYMND8 proteins from HEK293T cells overexpressing these proteins, followed by immunoprecipitation (IP) with anti‐HA antibody and immunoblotting (IB) analysis with anti‐Flag antibody. E) In vitro purified Flag‐tagged TBK1 protein as described in (D) was examined by coomassie blue staining (C.B.S). F) In vitro phosphorylation assay was performed by mixing in vitro purified Flag‐tagged TBK1 and HA‐tagged KDM5C proteins from HEK293T cells overexpressing these proteins, followed by immunoblotting (IB) analysis with antibodies as indicated. p‐TBK1: Anti‐TBK1 phosphorylated at serine 172 antibody. G) MCF7 cells were infected with lenti‐viral control shRNA (shCTL) or shRNA specific against KDM5C (shKDM5C) or ZYMND8 (shZYMND8) for three days, followed by immunoblotting analysis using antibodies as indicated. p‐IRF3: Anti‐IRF3 phosphorylated at serine 396 antibody; p‐STAT1: Anti‐STAT1 phosphorylated at tyrosine 701 antibody. Asterisk indicated non‐specific band. H,J) MCF7 cells were treated with or without C70 (5 μ m, 3 days) followed by immunoblotting (IB) analysis using antibodies as indicated (H) or RT‐qPCR analysis to examine the expression of selected genes as indicated (J). Asterisk indicated non‐specific band. I,K) MCF7 cells were transfected with siCTL or siKDM5C in the presence or absence of control vector, KDM5C (WT)‐R or KDM5C (H514A)‐R for three days, and then treated with or without estrogen (E₂, 10⁻⁷ m, 6 h) followed by immunoblotting (IB) analysis using antibodies as indicated (I) or RT‐qPCR analysis to examine the expression of selected genes as indicated (K). Asterisk indicated non‐specific band. L) MCF7 cells cultured in the presence of estrogen (E₂, 10⁻⁷ m) were treated with or without fulvestrant (ICI, 1 μ m for MCF7, 0.1 μ m for T47D), C70 (5 μ m), or C48 (10 μ m) alone or in combination for duration as indicated followed by cell proliferation assay (± SD, ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001). M) Xenograft experiments were performed by injecting MCF7 cells subcutaneously into female BALB/C nude mice and then treated with estrogen (E₂) in the presence of fulvestrant (ICI) or C48 alone or in combination. Tumors were then excised, photographed, and weighted four weeks after subcutaneous injection (± SD, ^** p < 0.01).