Autocrine/paracrine production of IFN-β by NF-κB contributes to HAPLN1 matrikine–induced STAT1 activation. (A) Representative western blot analysis of indicated proteins in RPMI8226 cells pretreated with IKK16 (10 μM) or dimethyl sulfoxide (DMSO) for 10 minutes and stimulated with 100 nM of MBP or MBP-PTR1 for 6 hours. (B) Representative western blot analysis of indicated proteins in RPMI8226 cells pretreated for 10 minutes with cycloheximide (CHX; 20 μg/mL) or brefeldin A (BFA; 3 μg/mL) and stimulated with 100 nM of MBP or MBP-PTR1 for 6 hours. (C) RPMI8226 cells were stimulated with 100 nM of MBP or MBP-PTR1 for 6 hours (lane 1 and 2). RPMI8226 cells were stimulated with 100 nM of MBP or MBP-PTR1 for 5 hours and subsequently washed with fresh media. Conditioned medium (CM) was harvested after a 1-hour incubation of fresh media without MBP or MBP-PTR1. CM was added to fresh RPMI8226 cells for 15 minutes (lane 3 and 4) and analyzed using western blotting for the indicated proteins. (D) RPMI8226 (WT and STAT1 KO) cells were stimulated with 100 nM of MBP or MBP-PTR1 for 6 hours. The IFNB1 mRNA level was quantified using qRT-PCR and normalized to GAPDH and fold change relative to control (MBP in WT cells) was plotted. (E) RPMI8226 cells were treated as in panel A. The mRNA level of IFNB1 was quantified using qRT-PCR and normalized to GAPDH and fold change relative to control (MBP and DMSO treated cells) was plotted. (F) Representative western blot analysis of indicated proteins in RPMI8226 cells pretreated with 10 μg/mL of anifrolumab or human immunoglobulin G (IgG) for 10 minutes and stimulated with 100 nM of MBP or MBP-PTR1 for 6 hours or IFN-β (50 pg/mL) for 15 minutes. The graph represents the means ± SEM of 3 biological replicates for panels D-E, each performed in duplicates. ∗P < .05, ∗∗P < .01.