Fig. 3: Myeloma TP/2DDR enhances CIITA expression in osteocytes via the STAT1/IRF1 signaling pathway.
From: Osteocyte CIITA aggravates osteolytic bone lesions in myeloma
a–b The levels of CIITA mRNA (a, n = 3 biological replicates) or protein (b) in MLO-Y4 or MLO-A5 osteocytes co-cultured with the high TP–expressing ARP-1 cells carrying non-targeted control shRNAs (shCtrl) or TP shRNAs (shTP) or co-cultured with low TP–expressing MM.1 S cells carrying empty control vector (Vec) or TP cDNAs (TP). c–e Levels of CIITA mRNA (c, n = 3 biological replicates) and protein (d) as well as phosphorylated (p) or non-phosphorylated ERK1/2, Akt, Syk, STAT1, and JAK1 (e) in MLO-Y4 and MLO-A5 cells cultured with 2DDR. (GAPDH: protein loading controls). f Levels of the p-STAT1 or STAT1 in primary osteocytes cultured with the CM of shCtrl or shTP ARP-1 cells or with the CM of Vec or TP MM.1 S cells. g–h Levels of p-SyK, Syk, p-STAT1, or STAT1 (g), Ciita, Tfsf11, and Sost mRNAs (h, n = 3 biological replicates) in MLO-Y4 or MLO-A5 cells cultured with 2DDR, IgG control, or anti-αv antibody (Ab). i Levels of p-STAT1 in whole-cell lysates or cytosolic or nuclear fractions of MLO-Y4 and MLO-A5 cells cultured without or with 2DDR. Loading controls: GAPDH for cytosol fractions (Cyt); H3 for nuclear fractions (Nuc). WCL, whole cell lysate. j The enrichment of p-STAT1 in the promoter of Irf1 gene in MLO-Y4 and MLO-A5 cells cultured with 2DDR. IgG levels served as controls. k The protein levels of IRF1 in MLO-Y4 and MLO-A5 cells cultured with 2DDR. l Co-immunoprecipitation of p-STAT1 with IRF1 in MLO-Y4 and MLO-A5 cells cultured with 2DDR. m The enrichment of p-STAT1 in the promoter of Ciita gene in MLO-Y4 and MLO-A5 cells cultured with 2DDR (n = 3 biological replicates). n–o The relative expression of Ciita (n), Tnfsf11 and Sost (o) mRNA in MLO-Y4 and MLO-A5 cells cultured with 2DDR in the absence or presence of STAT1 inhibitor fludarabine (Flu; 10 µM) (n = 3 biological replicates). b, d, e–g, i, k, and l are representative of two independent experiments. Data are mean ± SD. P values were determined using one-way ANOVA with Tukey’s multiple comparisons test.
