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. 2022 May 24;14(11):2580. doi: 10.3390/cancers14112580

Figure 5.

Figure 5

MUC1-C forms a nuclear complex with STAT1 and IRF1 in activating RIG-I, MDA5 and STING. (A) BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days and stimulated with IFN-β for 4 h were analyzed for STAT1 and IRF1 mRNA levels by qRT-PCR (left). The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). (B) Nuclear lysates from BT-549 cells stimulated with IFN-β for 4 h were precipitated with anti-MUC1-C and a control IgG. Input lysate and the precipitates were immunoblotted with antibodies against the indicated proteins. (C) BT-549 cells expressing a control CshRNA, STAT1shRNA or IRF1shRNA were analyzed for the indicated mRNA levels by qRT-PCR. The results (mean ± SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained for the CshRNA cells (assigned a value of 1). (D–F) Schemas of the RIG-I (D), MDA5 (E) and STING (F) promoter regions with highlighting localization of ISREs, IREs and GAS. Soluble chromatin from BT-549/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days was precipitated with a control IgG, anti-MUC1-C, anti-STAT1 and anti-IRF1. The DNA samples were amplified by qPCR with primers for the respective promoter regions. The results (mean ± SD of 3 determinations) are expressed as fold enrichment relative to that obtained with the IgG control (assigned a value of 1). The asterisk (*) denotes a p-value < 0.05. Uncropped Western Blots can be found at supplemental original images.