Figure 5. Inhibition of SETD1B diminishes H3K4me3 level and iNOS expression in MDSCs in tumor-bearing mice.

A. Chaetocin was tested in a 10-dose IC50 mode with 3-fold serial dilutions using [³H]S-adenosyl-methionine as a substrate with recombinant human HMTases as indicated. The enzyme activity was then analyzed and plotted against chaetocin concentrations. IC₅₀ was calculated using the GraphPad Prism program. B. 4T1 tumor cells were injected s.c. into the mammary glands of BALB/c mice. Tumor-bearing mice were treated i.p. 9 (left panel, small tumor group) and 21 (right panel, large tumor group) days after tumor cell injection with solvent control (9 day tumor-bearing mice: n=9. 21 day tumor-bearing mice: n=4) or chaetocin (9 day tumor-bearing mice: n=9. 21 day tumor-bearing mice: n=4) daily for 7 days. Shown is the quantification of tumor sizes before and after chaetocin treatment. C. Spleens were collected from control and chaetocin-treated tumor-bearing mice from the large tumor group. CD11b⁺Gr1⁺ cells were isolated from the spleens using CD11b Microbeads and LS columns. Purity of the isolated cells was determined by staining cells with IgG or CD11b- and Gr1-specific mAbs and flow cytometry analysis. Shown are representative images of IgG isotype control and CD11b-/Gr1-specific mAb staining of the purified cells from control mice (top panel) and chaetocin-treated mice (bottom panel). D. Comparison of MDSC purity from control and chaetocin-treated mice as shown in C. E. The purified MDSCs from control and chaetocin-treated mice as shown in C were analyzed by Western blotting for total cellular H3K4me3 levels. β-actin was used as normalization control. F. MDSCs were purified from spleens of control (n=3) and chaetocin-treated (n=3) tumor-bearing mice with large tumors and analyzed for iNOS mRNA level by qPCR.