Extended Data Figure 8. MARCH-1 dependent internalization of HLA-DR in CD14⁺ monocytes in myeloma environment.

(A) Gating strategy for HLA-DR on CD14⁺ cells. (B) Knockdown of MARCH-1 by siRNA rescues presentation of HLA-DR molecules on the surface of CD14⁺ monocytes co-cultured with MM cells. Representative FACS profiles show higher numbers of HLA-DR⁺ cells after MARCH-1 knockdown with siRNA (experiment performed 3 times with 3 different donors using 2 different cell lines/2 different siRNA for MARCH-1, non-targeting si-RNA is used as a control). (C) Mean fluorescence intensity demonstrates the two to 4.5-fold increase in the levels of HLA-DR protein expression on the cell surface of CD14⁺ cells. (D) qPCR data for relative expression of MARCH-1 in CD14⁺ cells after siRNA knockdown as compared to the si-RNA control. Assay performed twice with independent donors/2 cell lines/2 siRNAs, performed in triplicates. Representative data from one experiment is shown. In MM1.S cells, siRNA knockdown leads to reduction of MARCH-1 expression to 0.46 and 0.32 (median value, amplified with primer pair 2) and to 0.40 and 0.26 (median value, amplified with primer pair 3) as compared to control siRNA (median value, 1.0). In OPM2 cells, siRNA knockdown leads to reduction of MARCH-1 expression to 0.65 and 0.7 (median value, amplified with primer pair 2) and to 0.29 and 0.36 (median value, amplified with primer pair 3) as compared to control siRNA (median value, 1.0). Bars represent STDEVP. (E) CD14⁺ cells with lower levels of MARCH-1 have increased HLA-DR protein on their cell surface. Experiment performed twice with two independent donors/2 cell lines/2 siRNAs. (F) Stronger correlation of DAPI and HLA-DR localization in MM1.S and OPM2 cells in control cells as compared to those after MARCH-1-siRNA transfection. Experiment performed twice with two independent donors/2 cell lines/2 siRNAs.