Silencing of March1 by siRNA.

Splenocytes from tumor-bearing C57BL/6 mice were incubated with biotin anti-mouse Ly6C (1:200; HK1.4; Biolegend) followed by streptavidin microbeads (Miltenyi Biotec), and then Ly6C⁺ cells were positively selected on a magnetic field according to the manufacturer’s instructions (MACS separation columns MS, Miltenyi Biotec). Ly6C⁺ cells were seeded in 12-well plates (1.5 × 10⁶), cultured with 250 ng/ml LPS, and transfected with 20 μl (10 μΜ) of either March1 siRNA cocktail (Santa Cruz Biotechnology, sc-106199) or scramble si-RNA sequence (Santa Cruz Biotechnology, sc-37007) using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) according to the manufacturer’s protocol. Forty-eight hours after transfection, cells were washed, harvested, and tested with qPCR for knockdown efficiency. The expression of MHC II in transfected cells was assessed with FACS. Transfected Ly6C⁺ cells (4 × 10⁵), with either scramble or si-March1, were s.c. coinjected with 3 × 10⁵ B16-F10 cells into C57BL/6 mice. The tumor volume was monitored from day 7 to day 12.