Fig. 6: Mogat1 inhibition promotes an inflamed tumor microenvironment.

a–c t-distributed stochastic neighbor embedding (t-SNE) plots visualizing single-cell flow cytometry data from shCTRL and shMogat1 MMTV-PyMT tumors. a Total immune and non-immune cell populations. b All cell populations. c Subsets of CD4⁺ and CD8⁺ T cells based on PD1 expression. The comprehensive flow cytometry gating strategy for these panels is presented in Supplementary Fig. 8a, b. d–g Quantification of immune cell populations in shCTRL and shMogat1 MMTV-PyMT tumors by flow cytometry. These quantitative data (d) for total immune cells (CD45⁺). e for total CD3⁺T cells and T cell sub populations. f for B, NK and DC cells. g for tumor-associated macrophages (TAMs, CD45⁺CD11b⁺F4/80⁺) and tumor-associated neutrophils (TANs, CD45⁺CD11b⁺Ly6G⁺), were all derived using the gating strategies presented in Supplementary Fig. 8a, b. n = 5 mice per groups. h Representative immunofluorescence images of shCTRL and shMogat1 MMTV-PyMT tumor sections stained for CD8 (red) to visualize cytotoxic T cell infiltration. Scale bar: 100 μm. i, j FACS analysis of the IFNγ, Granzyme B (i) and exhaustion marker TOX, CTLA-4 and LAG3 expressing CD8 T cells infiltrating MMTV-PyMT tumors. n = 5 mice per groups. In vitro cytotoxicity assay assessing the susceptibility of shCTRL or shMogat1 (k) MMTV-PyMT-OVA and (n) B16/F10-OVA tumor cells to CD8⁺ T cell-mediated killing. Quantification of tumor cell killing at various E:T ratios. (n = 4 biological cell cultures per group). IFNγ secretion by CD8⁺ T cells following co-culture with shCTRL or shMogat1 l MMTV-PyMT-OVA and o B16-OVA cells, as measured by ELISA. (n = 4 biological cell cultures per group). Flow cytometry analysis of the frequency of Granzyme B-positive (GraB⁺) CD8⁺ T cells after co-culture with shCTRL or shMogat1 (m) MMTV-PyMT-OVA and (p) B16-OVA cells. (n = 4 biological cell cultures per group in data m, n = 3 biological cell cultures per group in data p). q, r Schematic representation of a transwell assay involving in vitro activated OT1 CD8⁺ T cells combined with shCTRL or shMogat1 MMTV-PyMT-OVA tumor cells media. r CD8⁺ T Cell migration and PI⁺ dead cells were analyzed by transwell assay (n = 4 biological cell cultures in shCTRL group, n = 3 biological cell cultures in shMogat1 group). q is created in BioRender. https://BioRender.com/k35b440 (n = 4 biological cell cultures in shCTRL group, n = 3 biological cell cultures in shMogat1 group). Data (a–j) are representative of two independent experiments from 5 mice per group per experiment. Data (k–r) are representative of two independent experiments. Data are presented as mean ± SD. Statistical significance was calculated using unpaired Student’s t tests. ns, not significant. Source data are provided as a Source Data file.