Fig 2: Rab7 GTPase controls tumor stimulation of lal-/- MDSCs(A) Ly6G+ cells from the wild type or lal-/- bone marrow were transfected with control or Rab7 GTPase siRNA for 24 h, and 2 × 105 transfected Ly6G+ cells were co-cultured with B16 melanoma cells (2 × 104) or LLC cells (2 × 104) in vitro. Cells were counted 48 h later. Data were expressed as mean ± SD, n = 5. *P < 0.05, **P < 0.001; -, no transfection; C, transfected with control siRNA; Rab7, transfected with Rab7 siRNA. (B) Ly6G+ cells from the syngeneic C57BL/6 wild type or lal-/- bone marrow were transfected with control or Rab7 GTPase siRNA for 24 h. Transfected Ly6G+ cells were mixed with B16 melanoma cells (2 × 105) or LLC cells (5 × 105) (3:1), followed by subcutaneously flank injection into C57BL/6 wild type recipient mice. Tumor sizes were measured at post injection day 10. The average tumor size of each group and p values are marked with n = 10; (C) Ly6G+ cells from the allogeneic FVB/N wild type or lal-/- bone marrow were transfected with control or Rab7 GTPase siRNA for 24 h. Transfected Ly6G+ cells were mixed with B16 melanoma cells (2 × 105) or LLC cells (5 × 105) (3:1), followed by subcutaneously flank injection into the FVB/N wild type recipient mice. Tumor sizes were measured at post injection day 10. The average tumor size of each group and p values are marked with n = 12; (D) Ly6G+ cells from C57BL/6 wild type or lal-/- bone marrow were transfected with control or Rab7 GTPase siRNA for 24 h. Transfected Ly6G+ cells (2.5 × 106) were mixed with B16 melanoma cells (5 × 105) (5:1), followed by intravenously injection into C57BL/6 wild type recipient mice through tail vein. B16 melanoma colony numbers were counted at 2 weeks post injection. The average colony number of each group and p values are marked with n = 10; (E) Ly6G+ cells from C57BL/6 wild type or lal-/- bone marrow were transfected with control or Rab7 GTPase siRNA for 24 h. Transfected Ly6G+ cells (2.5 × 106) were mixed with LLC cells (5 × 105) (5:1), and intravenously injected into C57BL/6 wild type recipient mice through tail vein. Lung histology of LLC invasion was examined at 2 months post injection. The representative H & E sections were shown, n = 10 mice; (F) Ki67 staining of E. For A-F: -, no transfection; C, transfected with control siRNA; Rab7, transfected with Rab7 siRNA.

Fig 3: Rab7 GTPase controls ROS production and the mitochondrial membrane potential(A) Western blot analysis of Rab7 GTPase expression in wild type and lal-/- bone marrow Ly6G+ cells with control or Rab7 GTPase siRNA transfection for 2 d. Actin was used as loading control. Results are representative of three independent experiments; (B) ROS production in wild type and lal-/- bone marrow Ly6G+ cells, or in HD1A and HD1B myeloid cells with control or Rab7 GTPase siRNA transfection. ROS levels were measured by flow cytometry. Results are mean ± SD, n = 4, *p < 0.05, **p < 0.001; (C) The mitochondrial membrane potential in wild type and lal-/- bone marrow Ly6G+ cells, with control or Rab7 GTPase siRNA transfection. The mitochondrial membrane potential was measured using JC1 staining by flow cytometry. The results are mean from four independent experiments (n = 4), *p < 0.05, **p < 0.001; For A-C, -, no transfection; C, transfected with control siRNA; Rab7, transfected with Rab7 siRNA. (D) The mitochondrial membrane potential of HD1A and HD1B cells was measured by fluorescence microscope. Results are representative of three independent experiments, scale bar, 25 µm.

Fig 4: Analysis of cell populations following intraperitoneal challenge with A/WSN/1933 virus or UV-inactivated A/WSN/1933 virus. BALB/c mice (n = 5/group) were sacrificed at 12 h, 5, 7, and 14 days after intraperitoneal challenge with 5 × 106 pfu of A/WSN/1933 virus (WSN) or UV-inactivated A/WSN/1933 virus (UV-WSN). Peritoneal cells, bone marrow cells and splenocytes were harvested, counted, and stained with fluorescence-conjugated antibodies and analyzed by flow cytometry. Cells from control mice at 14 days after intraperitoneal injection of PBS were used as an uninfected control. (A-C) FSClowSSClow cells of peritoneal cells, bone marrow cells, splenocytes were gated, and lymphocyte populations of the peritoneal cavity (A), bone marrow (B), and spleen (C) were sorted into CD3 and CD19 subsets. (D,E) Peritoneal cells (FSClowSSChigh) were gated, and two populations, F4/80highCD11bhigh (macrophages) and F4/80lowCD11blow (non-macrophages) were analyzed. (D) Peritoneal macrophages (F4/80highCD11bhigh) were sorted for F4/80 and CD11b double-positive phenotype. (E) The dendritic cell and neutrophil populations of the peritoneal cavity were sorted from F4/80lowCD11blow population using CD11c and Ly6G markers. Right panel showed the indicated cell population (%) among FSClowSSClow cells (A-C) and FSClowSSChigh cells (D,E). Each figure is representative of three independent experiments. **p < 0.005, ***p < 0.0005.

Fig 5: The transcriptome of the pre-BCR-dependent expansion stage is governed by MYC-associated gene expression networks and requires repression of Ebf1 expression.a Highlighted population of the pre-BCR-dependent cluster (left) and feature plots for the pre-BCR-dependent markers highly expressed during this stage (right). b Violin plot of B-lymphoid transcription-factor expression, including Ebf1, Pax5, and Ikzf1 across B-cell development. c Violin plot of additional genes that are downregulated during pre-BCR-dependent proliferation, which includes Il7r, and EBF1-target genes such as Cd79a and Cd79b. d Landscape In silico Deletion Analysis (LISA) to predict the transcriptional regulator of the top 100 differentially upregulated genes during pre-BCR-dependent proliferation (left table). Gene-set enrichment analysis was performed to identify gene sets from the Molecular Signature Database that were enriched in the pre-BCR-dependent cells. Comparison between the pre-BCR-independent cells (left side) and the pre-BCR-dependent cells was performed (right side). Statistical significance was calculated using one-sided Wilcoxon rank-sum test. e EBF1-binding sites at the promoters of Nrgn, Slc7a5 and Slc3a2 with indicated MACS peak calls20. Data were obtained from GSM2863146. f Log2-transformed FPKM expression values obtained from RNA-seq of wild-type and Pax5+/- × Ebf1+/- (PE) leukemic progenitor B cells. Progenitor B cells were obtained via negative selection of CD11c, TER119, Ly6G, Ig Kappa, and Ig Lambda and positive selection of CD19. Statistical significance was determined using a two-tailed unpaired student t-test for Ybx3 (P = 0.0024) and Slc7a5 (P = 0.0001). RNA-seq data were obtained from GSE148680. A two-tailed Mann–Whitney test was performed for Slc3a2 (P = 0.0083) due to non-normal distribution. n = 7 biologically independent samples over one independent experiment. Color scales in a, b, and c represent natural log-transformed SCTransform-corrected counts.