Fig 2: Myeloma TP/2DDR enhances CIITA expression in osteocytes via the STAT1/IRF1 signaling pathway.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.

Fig 3: Interaction networks of the genes up- and downregulated by ATRA in the retinoid-sensitive cell-lines. The 754 genes whose up- or downregulation is proportional to ATRA-sensitivity were used to construct an interaction network based on the encoded proteins (STRING database, https://string-db.org). The 2 upregulated modules (MHC Class-I Loading and presentation; Interferon Response) can be further condensed into a single group, as indicated by a red circle. The single genes belonging to this module are indicated by red dots. The higher the average upregulation afforded by ATRA, the larger the size of each red dot. The 4 downregulated modules (Cell-cycle, Histone-cluster I, Chromatin-packaging/organization, DNA-repair) can be further clustered into a single group marked by a blue circle. The single genes belonging to this module are indicated by blue dots. The lower the fold-change observed following ATRA treatment, the larger the size of each blue dot. The heat-maps of the ATRA/DMSO fold changes observed for the genes belonging to the up- (right) and downregulated (left) interaction networks in each cell-line are shown. The cell-lines are ranked from left to right according to their decreasing sensitivity to ATRA (decreasing ATRA-score value). Luminal cell-lines are indicated in red, while basal cell-lines are marked in blue. The IRF1 and DTX3L mRNAs are marked with a red circle.

Fig 4: STAT1 modulates increased p65 recruitment to GAS/NFκB or ISRE/NFκB composite sites. (A) ChIP-PCR of STAT1 at Serpina3i, Steap4, Irf1, Ccl5, Ifit1, Gbp6, Cxcl10, and Gbp7 gene promoters (primers are listed in Table S2) in VSMC WT treated with IFNα (8 h), IFNγ (8 h), LPS (4 h), IFNα (8 h) + LPS (4 h), IFNγ (8 h) + LPS (4 h). Mean ± s.e.m., n = 2. (B) ChIP-PCR of p65 at Serpina3i, Steap4, Irf1, Ccl5, Ifit1, Gbp6, Cxcl10, and Gbp7 gene promoters in VSMC WT treated with IFNα (8 h), IFNγ (8 h), LPS (4 h), IFNα (8 h) + LPS (4 h), IFNγ (8 h) + LPS (4 h). Mean ± s.e.m., n = 2. (C) ChIP-PCR of p65 at Serpina3i, Steap4, Irf1, Ccl5, Ifit1, Gbp6, Cxcl10, and Gbp7 gene promoters in VSMC STAT1 KO treated with IFNα (8 h), IFNγ (8 h), LPS (4 h), IFNα (8 h) + LPS (4 h), IFNγ (8 h) + LPS (4 h). Mean ± s.e.m., n = 2.

Fig 5: Knockdown of IRF1 inhibited CD47 up-regulation induced by IFN-γ.(a) The protein expression of IRF1 and CD47 was analyzed by western blot analysis after knockdown of IRF1 by siRNA in A549 and NCI-H1975 cells. (b) Surface expression of CD47 was detected by flow cytometry after silenced IRF1 in two lung cancer cell lines. (c) SIRPα binding assay was presented to showed CD47 binding ability to SIRPα after knockdown of IRF1 in A549 cells. (d) Co-culture A549 cells with BMDMs, the phagocytosis index change was measured based on flow cytometry result. *, P < 0.05; **, P < 0.01.