Fig. 3

OSM overexpression represses endogenous IFN-β mRNA/ISGs and drives mesenchymal/CSC properties in TNBC-BT549 cells. a Endogenous IFN-β mRNA expression is repressed in BT549-OSM cells, as demonstrated by qRT-PCR (***P < 0.001), ± SEM, n = 3. b ISGs including STAT1, STAT2, IRF9, SOCS1, IRF1, IFI16, MX1, and OAS1 are repressed in BT549-OSM cells relative to BT549-GFP cells, as demonstrated by qRT-PCR (***P < 0.001, *****P < 0.00001), ± SEM, n = 3. c IFN-β signaling effectors including phosphorylated and total protein expression of STAT1, STAT2, and IRF9 are repressed in BT549-OSM cells relative to BT549-GFP cells (demonstrating repressed P-ISGF3), while OSM signaling effectors, including the expression of phosphorylated and total STAT3 and ERK1/2, are elevated in BT549-OSM cells, as demonstrated by Western analysis. The lines indicate separate Western blots using matched samples. d BT549-OSM cells have significantly increased tumor sphere initiation capacity (stem cell frequencies: 1:11 in BT549-OSM, 1:Inf (Infinity) in BT549-GFP; **P < 0.01), ± SD, n = 5. e BT549-OSM cells have robust tumor initiation capacity in vivo following 3 weeks of engraftment, relative to BT549-GFP cells (bioluminescent images and table showing tumor initiation frequencies: 1:Inf (Infinity) in BT549-GFP, 1:17,281 in BT549-OSM; *P = 0.05) ± SD, n = 5 mice. f BT549-OSM cells have enhanced migratory capacity relative to BT549-GFP cells (post-80 h; ***P < 0.001). g Elevated expression of an experimentally derived OSM target gene signature (top 20 induced genes) and low expression of an experimentally derived IFN-β target gene signature (top 20 induced genes) corresponds with the decreased patient survival in TNBC (red graph) compared to low expression of the OSM target gene signature and high expression of the IFN-β target gene signature (black graph) (P = 0.0031). h Sustained exogenous, recombinant IFN-β treatment (100 IU/mL; every 48 h for up to 6 weeks) is sufficient to restore canonical IFN-β-mediated P-ISGF3 signaling, with robust phosphorylation of STAT1 and STAT2 and increased expression of STAT1, STAT2, and IRF9 proteins and repressed expression of SNAIL. The lines indicate separate Western blots using matched samples. i Sustained exogenous recombinant IFN-β treatment (100 IU/mL; every 48 h for up to 6 weeks) significantly represses tumor sphere initiation capacity in BT549-OSM cells (stem cell frequencies: 1:90 in BT549-GFP, 1:5 in BT549-OSM, 1:44 in BT549-OSM + rec IFN-β; **P < 0.01; ***P < 0.001) ± SD, n = 6