Figure 6.

Loss of YTH N⁶-methyladenosine RNA binding protein 1 (Ythdf1) promotes reduction of myeloid-derived suppressor cells (MDSCs) by decreasing CXCL1 secretion. (A) Multiplex mouse cytokine immunoassay assessing levels of 23 cytokines in cell culture supernatants from MC38 and CT26 cell culture, and tumour lysates and sera from MC38 and CT26 syngeneic tumour models. (B) ELISA validation of levels of CXCL1 in the indicated materials as in (A) (n=4 each group). (C) MDSC migration was assessed in vitro with transwell migration assay using conditional medium derived from the indicated cells. CXCR2 inhibitor SB265610 was used at 5 µM. (D) RT-qPCR determination of Cxcl1 mRNA expression in the indicated cells. (E) Flow chart of T cell suppression assay. (F) Representative images of flow cytometry assessing the proliferation of CFSE-labelled T cells cocultured with MDSCs isolated as (E). (G) Quantification of (F) (two tailed t-test (C, D, E, G)).