Extended Data Fig. 7: Pathogenic IgG activates CXCR4/SDF1α axis through NF-κB pathway via binding with HSPA4.
(a) mRNA expression of Cxcr4 in tumor cells pretreated with PDTC, SB203580, SP600125, PD98059, or wortmannin and then stimulated by pathogenic immunoglobulin for 24 h (n = 5 biologically independent samples). (b) CD16/32 expression of 4T1, EMT6 tumor cells and macrophages (positive control) were analyzed by FACS. (c) NF-κB luciferase activation was measured using Dual-Luciferase Reporter Assay system 24 h after transfection (n = 5 biologically independent samples). (d) Binding of whole lysates of 4T1 tumor cells by ctrl or tumor immunoglobulin was detected by western blot. (e) The list of candidate proteins for antigen screening from mass spectrometry analysis. (f) NF-κB luciferase activation was measured using Dual-Luciferase Reporter Assay system 24 h after transfection with or without silence of candidate genes (n = 5 biologically independent samples). (g) mRNA expression of Hspa4, Ncl, Itgb3 and Itgb5 in tumor cells (n = 6 biologically independent samples). (h) HSPA4-Flag and Myc-tagged Ncl, Itgb3, Itgb5 were transfected into tumor cells. Then HSPA4-Flag was immunoprecipitated and other proteins were blotted by anti-Myc antibody. (i) Flag-tagged Hspa4 and Itgb5 were transfected into 4T1 cells. These proteins were immunoprecipitated (IP) with anti-flag antibody and immunoblotted (IB) with normal or tumor immunoglobulin. (j) Immunoblot analysis of HSPA4 level in HSPA4 knock-out (KO) (j) or ITGB5 level in ITGB5−KO (k) tumor cells. (l) Different N-glycosylation sites in the peptide of HSPA4 purified from 4T1, B16/F10 and Hepa 1-6 cells are characterized by mass spectrometry. Red color, underscore, italic represents N-glycosylation sites in 4T1, B16/F10 and Hepa 1-6 cells respectively. (m) λ chain of IgG1, IgG2a, IgG2b, IgG3, IgA, IgM and IgE from the serum of WT or two HSPA4-KO clone bearing mice (n = 8 biologically independent animals) were detected by CBA. (n) Representative immunohistochemistry images of CXCR4, and PGE2 expression in tumors with or without HSPA4 knock out. Scale bars, 50 μm. In all panels, data are plotted as mean +/− s.d. and analyzed using unpaired two-tailed Student’s t-tests. Data are representative of three (a-k, m,n) independent replicates.
