(A) Phospho-STAT3 (p-STAT3) levels from PMN-MDSCs from shControl or shCCL2 tumors measured by flow cytometry. Mean ± SEM. **p < 0.001. Unpaired, two-tailed Student's t test.
(B) Expression of C/ebpβ measured by qRT-PCR with data normalized to actin expression (mean relative expression ± SEM).
(C–H) Analysis of sorted intratumoral PMN-MDSCs from the mice with shControl tumors followed by treatment with a STAT3 inhibitor (S3I-201) (50 μM) for 18 hr. Phospho-STAT3 (p-STAT3) levels (C) and ROS levels (DCFDA staining) (D), gp91phox (E) S100A8 (F), S100A9 (G) and iNOS (H) expression levels were measured. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 (paired, two-tailed Student's t test).
(I–L) Sorted intratumoral PMN-MDSCs from shCCL2 tumor-bearing mice were treated with S31-201 and then coincubated with CD4+ T cells or CD8+ T cells. T cell proliferation assays: CD4+ T cells (I) and CD8+ T cells (J) are shown. Division index represents T cell divisions. *p < 0.05, **p < 0.01 (paired, two-tailed Student's t test). ζ chain expression of CD4+ T cells (K) and nitrotyrosine levels of CD8+ T cells (L) measured by flow cytometry. All data represent two or three independent experiments. *p < 0.05 (paired, two-tailed Student's t test). See also Figure S6.