Fig 2: Becn1 deficiency impairs homeostasis of the blood system. (A) Kaplan–Meier survival plot depicts survival curves for Becn1vKO mice (n = 53) and WT mice (n = 37). (B and C) These histograms exhibit the complete blood cell counts of peripheral blood samples from WT and Becn1vKO mice, including RBC (red blood cell), WBC (white blood cell), Neu (neutrophil), Lym (lymphoid cell), PLT (platelet) (B), HGB (hemoglobin), MCV (mean corpuscular volume), RDW (red cell distribution width) (C). Data are shown as mean ± SD, n = 5 mice per group. (D and E) This histogram shows the lineage distribution of peripheral blood (D) and bone marrow (E) for WT and Becn1vKO mice, including T cells (CD3+), B cells (B220+), and myeloid cells (CD11b+). Data are shown as mean ± SD, n = 5 mice per group. (F and G) The histograms display the number of HSPCs per million bone marrow cells of WT and Becn1vKO mice, including LT-HSC (long term-HSC, CD34-/Flt3-LSK), ST-HSC (short term-HSC, CD34+/Flt3-LSK), LSK (Lin-/Sca1+/c-Kit+) (F), MPP (multipotent progenitor, CD34+/Flt3+/LSK), CLP (common lymphoid progenitor, CD127+/Flt3+/LSlowKlow), CMP (common myeloid progenitor, Lin-/Sca1-/c-Kit+/CD34+/CD16/32low), GMP (granulocyte/macrophage progenitor, Lin-/c-Kit+/Sca1-/CD34+/CD16/32high) and MEP (megakaryocytic/erythroid progenitor, Lin-/c-Kit+/Sca1-/CD34-/CD16/32-) (G) (n = 5 mice per group). Data are shown as mean ± SD. (H) The histogram displays the number of LT-HSC and ST-HSC per femur (n = 5 mice per group). Data are shown as mean ± SD. (I) This photograph (left) and scatter plot (right) exhibit the differences of spleens from WT and Becn1vKO mice (n = 3 for each group). Data are shown as mean ± SD. (J and K) The histograms display the ratio (J) and absolute number (K) of HSPCs from spleens of WT and Becn1vKO mice. Data are shown as mean ± SD, n = 3 mice for WT and 4 mice for Becn1vKO group. The gating strategy of HSPCs is shown in sup. Fig. 1A 1A for (F–H) and in sup. Fig. 1E for (J and K). Adult mice were analyzed at 4 to 5-month-old for (B–K).

Fig 3: LSD1 depletion results in sustained tumor infiltration of CD8+ T cells.a, b Frequencies of tumor-infiltrating CD3+CD4+ T cells, CD3+CD8+ T cells, CD3-CD49b+ natural killer (NK) cells and CD11b+Gr1+ myeloid-derived suppressor cells (MDSCs) among gated CD45+ leukocytes (a) and cell numbers per gram of tumor (b) analyzed by flow cytometry on day 12 and 18 after MC38 tumor implantation in Lsd1f/f and Cd4-Cre+Lsd1f/f mice (Lsd1f/f d12 group, n = 5; Cd4-Cre+Lsd1f/f d12 group, n = 5; Lsd1f/f d18 group, n = 4; Cd4-Cre+Lsd1f/f d18 group, n = 5). c Calculated ratios of intratumoral CD8+ T cell number over MDSCs (n = 5, 5, 4, or 5, respectively). d–f Representative flow plots (d), percentages (e) and cell numbers (f) of CD8+Tetramer+ TILs in implanted MC38-OVA tumors analyzed by flow cytometry on day 18 (Lsd1f/f group, n = 14; Cd4-Cre+Lsd1f/f group, n = 10). g–i Frequencies of Ki-67-expressing CD8+ TILs (g), frequencies (h) and mean fluorescence intensity (MFI, i) of GzmB-expressing CD8+ TILs analyzed by flow cytometry on day 18 after MC38 tumor implantation (n = 4 per group). j IFN-? expression by MC38-OVA tumor-infiltrating CD8+ TILs analyzed by flow cytometry after stimulation with OVA257-264 peptides or PMA/Ionomycin (P/I) in the presence of GolgiPlug (n = 4 per group). Data represent two independent experiments (a–c, g–j) or are pooled from three independent experiments (e, f) and are presented as mean ± SEM (a–j). Each dot on the graphs represents an individual mouse. Sample sizes are as indicated. Statistical significance was determined by two-sided unpaired t test; ns, not significant (a–j). Source data are provided as a Source data file.

Fig 4: Impaired leukocyte recruitment in response to TNF-a–induced peritonitis in STING–/– mice compared with WT mice.(A–H) WT and STING–/– mice received PBS, TNF-a 4 hours (h), or TNF-a 24 h (A–D) or thioglycollate 3 days (E–H) via i.p. injection, and the peritoneal lavage was analyzed by FACS. (A) Representative flow cytometric panels of CD45+CD4+ cells recruited to the peritoneal cavity at 24 h and of CD45+Gr1+ at 4 h. (B–D) Quantification of total CD45+ (B), CD45+CD4+ (C), and CD45+Gr1+ (D) recruited to the peritoneal cavity. n = 3 (control); n = 12 (TNF-a 24 h); n = 3-4 (TNF-a 4 h). (E) Representative flow cytometric panels of CD45+CD11b+ cells recruited to the peritoneal cavity in response to thioglycolate of 3 independent experiments. (F–H) Quantification of CD45+ (F), CD45+CD11b+ (G), and CD45+CD4+ (H) recruited cells to the peritoneal cavity. n = 3 (control); n = 8 (thioglycollate). Data are shown as mean ± SEM values. *P < 0.05 and **P < 0.01; 2-way ANOVA (B–D) and 1-way ANOVA (F–H).

Fig 5: Asthma exacerbation is alleviated in macrophage-Arf6 cKO mice.WT and macrophage-Arf6 cKO mice were immunized by intraperitoneal injection of OVA with aluminium hydroxide as an adjuvant once per week for 3 weeks. At days 7, 10, and 13 after the last immunization, the mice received intranasal injection of OVA. (A) At day 1 after the last OVA challenge, the number of each indicated leukocyte subset in BALF obtained from WT and macrophage-Arf6 cKO (Arf6 cKO) mice was examined by FACS (n = 3 mice per group; mean ± SD are shown). ***P < 0.001, 1-way ANOVA with Tukey’s test for CD45.2-positive cells. (B) The number of Siglec-F–positive granulocytes in BALF obtained from WT and macrophage-Arf6 cKO mice was examined by FACS (n = 5 mice per group). The combined results from 2 independent experiments are shown. ***P < 0.001; 1-way ANOVA with Tukey’s test. (C) Lung tissue sections were stained with PAS-hematoxylin at day 1 after the last OVA challenge. Data are representative of 3 independent experiments. Scale bar: 150 µm (top); 20 µm (bottom). (D) The number of Siglec-F–positive granulocytes in BALF obtained from WT and macrophage-Arf6 cKO mice challenged with HDM was examined by FACS (n = 5 mice per group). ***P < 0.001, 2-tailed Student’s t test. (E–H) The amount of OVA-specific IgG1 in serum (E), OVA-specific IgE in BALF (F), IL-5 in BALF (G), and IL-13 in BALF (H) obtained from the indicated mice was examined by ELISA (n = 5–14 mice per group). Each symbol represents 1 mouse. The combined results from 2 independent experiments are shown. ***P < 0.001, 2-tailed Student’s t test.