Fig 2: Hematopoiesis-specific Cited2 deletion does not derail multilineage hematopoiesis(A) Cited2 mRNA levels in cells isolated from 8-week-old C57BL/6 mice (n = 4).(B) Cited2 expression in different hematopoietic compartments determined using single-cell SMART2-seq.(C) Cited2 expression in HSCs and committed progenitor cell compartments determined by 10× Genomics single-cell RNA-seq.(D) Cited2fl/fl mice were bred to Vav-iCre mice to generate Cited2fl/fl;Vav-iCre (Cited2CKO) mice. Cited2fl/fl mice were used as controls (Cited2CTL).(E) Cited2 expression in c-Kit+ cells from BM of Cited2CTL and Cited2CKO mice (n = 4).(F) PB counts of 8- to 10-week-old mice (n = 14).(G) Total BM cellularity (n = 6–9).(H) Total number of differentiated myeloid (Gr-1+Mac-1+) and B cells (CD19+B220+) in BM and spleen (Spl) (n = 6–9).(I) Total number of mature CD4+ and CD8+ T cells in spleens (n = 6–9).(J) Total number of Lin- cells, LK cells, pre-GM (LKCD41-Fc?RII/III-CD150-CD105-), GMP (LKCD41-Fc?RII/III+), Pre-MegE (LKCD41-Fc?RII/III-CD150-CD105+), MkP (LKCD41+CD150+) cells (n = 5).(K) Total number of Pre-CFU-E (LKCD41-Fc?RII/III-CD150+CD105+), CFU-E (LKCD41-Fc?RII/III-CD150+CD105+CD71+Ter119-), and Pro Ery (LKCD41-Fc?RII/III-CD150+CD105+CD71+Ter119+) cells (n = 5).(L) Colony-forming unit (CFU) assay performed with 104 BM cells from 8- to 10-week-old mice. CFU-megakaryocyte (CFU-Mk), CFU-erythroid (CFU-E), burst-forming unit (BFU-E), CFU-granulocyte (CFU-G), CFU-monocyte/macrophage (CFU-M), CFU-granulocyte, and monocyte/macrophage (CFU-GM); at least three lineages (CFU-Mix), CFU with all four lineages, granulocyte, erythroid, monocyte/macrophage, and megakaryocyte (CFU-GEMM) (n = 7).(M) Colony counts of primary and secondary cultures (n = 7). For (B), (C), and (F)–(M), data are mean ± SEM. * p < 0.05, **p < 0.01

Fig 3: Inulin Supplementation Overcomes Melanoma Resistance to MEKi(A) Growth of NRASQ61K mouse melanoma cells (1 × 106) (control, n = 11; mucin, n = 9; inulin, n = 9; MEKi, n = 10; MEKi+mucin, n = 8; MEKi+inulin, n = 10) that were subcutaneously transplanted in syngeneic C57BL/6 mice that received control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation. When tumors reached a volume of 10–20 mm2, mice were administered MEKi (PD325901, 10 mg/kg) once daily by gavage. Tumor volume was assessed every 4 days.(B) Number of tumor-infiltrating effector (CD44hi) CD4+ and CD8+ T cells and total CD45+ cells per tumor weight (in grams) in mice treated as in (A) (MEKi, n = 7; MEKi+mucin, n = 8; MEKi+inulin, n = 8).(C) Number of tumor-infiltrating DCs and DC subsets per tumor weight (in grams) and expression of MHC class I on DCs in mice treated as in (A) (MEKi, n = 8; MEKi+mucin, n = 7; MEKi+inulin, n = 8).(D) Pie chart of taxa enriched in inulin-treated mice and negatively correlated with MaN-RAS tumor size (n = 10).(E) Pie chart of taxa enriched in mucin-treated mice microbiota that negatively correlate with MaN-RAS tumor size (n = 10).(F) Relative abundance of taxa enriched in inulin+MEKi-treated mice and negatively correlated with MaN-RAS tumor size (n = 10).(G) Relative abundance of taxa enriched in mucin+MEKi-treated mice and negatively correlated with MaN-RAS tumor size (n = 10).Data are representative of two independent experiments. Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s correction (B and C) or by two-way ANOVA with Bonferroni’s correction (A).

Fig 4: Administration of Mucin or Inulin Reduces Tumor Growth and Induces Anti-tumor Immunity(A) Growth of YUMM1.5 tumors that were subcutaneously transplanted in syngeneic C57BL/6 mice. Mice were provided with a control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation (control, n = 12; mucin, n = 15; inulin, n = 15).(B) Quantification of tumor-infiltrating total CD45+ cells and effector (CD44hi) CD4+ or CD8+ T cells from mice treated as in (A) (control, n = 9; mucin, n = 10; inulin, n = 10).(C) Quantification of tumor-infiltrating, IFN-?-producing CD4+ T cells from mice treated as in (A) (n = 10).(D) Quantification of tumor-infiltrating total DCs and DC subsets in mice treated as in (A) (control, n = 9; mucin, n = 10; inulin, n = 10).(E) MFI of MHC class I and MHC class II on tumor-infiltrating DCs in mice treated as in (A) (control, n = 9; mucin, n = 10; inulin, n = 10).(F) Wild-type C57BL/6 mice (n = 12) were fed control or 15% inulin-supplemented chow starting 14 days before subcutaneous (s.c.) injection of YUMM1.5 melanoma cells (1 × 106). Anti-mouse Thy1.2 or control immunoglobulin G (IgG, 400 µg) were injected two times a week starting 3 days after tumor inoculation (n = 12). Tumor volume was assessed two times a week. FACS analysis revealed >90% depletion of blood CD4+ and CD8+ T cells on day 8 after tumor inoculation. Data are representative of three independent experiments (A–E) or one experiment (F). Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Bonferroni’s correction (A and F) or by one-way ANOVA with Bonferroni’s correction (B–E).

Fig 5: Mucin and Inulin Induce Enhanced Expression of Immunoregulatory Genes in Tumors(A) qPCR analysis of immune-related genes in subcutaneously transplanted melanoma grown in syngeneic C57BL/6 mice that received a control diet, 3% mucin in drinking water, or 15% inulin-supplemented chow starting 14 days before tumor inoculation (n = 6).(B) Quantification of CD45.1+ OT-I CD8+ T cells in the tumor-draining lymph nodes (TdLN) and non-draining lymph nodes (ndLN) of C57BL/6 mice (CD45.2+) treated with or without mucin and injected with B16-OVA melanoma cells (TdLN, n = 7; ndLN, n = 8). Right dot plots show gating of CD45.1+ CD8+ cells.(C) Serum cytokine and chemokine levels in naive WT mice treated with or without mucin (n = 10).(D) Serum cytokines in WT mice treated with or without mucin on day 10 after tumor inoculation (n = 10).Data are representative of three independent experiments (A and B) or one experiment (C and D). Graphs show the mean ± SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s correction (A) or by two-tailed t test or Mann-Whitney U test (B–D).