Fig 1: CBFβ regulates AM proliferation and self-renewal(A) Cell cycle analysis of AMs from the young hosts using list of genes associated with S or G2/M phases of cell cycle. UMAP (left) and quantification (right) of the cells predicted to be in each cell-cycle phase with respect clustering shown in Figure 1B.(B) Dot plot revealing the score for S-Phase or G/2M-Phase related genes, transcription factors related to proliferation and identity of AMs, and transcription factors associated with the suppression of the ESC-like feature of AMs.(C) Percentage of Ki-67 expressing macrophages in response to its major growth factor culture on treatment with different concentrations of Ro 5–3335 (100μM, 50μM, and 0 μM respectively, each dot represents one biological replicate).(D) Adherent cells in BAL from CbfbΔLysm (n = 3) or wild-type (n = 3) mice were stimulated with 10 ng/ml GM-CSF in vitro. The representative FACS plot (left) and quantification (right) of Ki-67 expressing AMs following 24 h in culture. (E to G) Bone-marrow-chimeric mice were constructed by transferring bone marrow from CbfbΔLysm or wild-type (WT) mice to age and gender matched WT mice (each dot represents one mouse).(E) Representative FACS plot (left) and quantification (right) of AMs in the lungs of CbfbΔLysm or wild-type BM chimeric mice.(F) Geometry mean fluorescence intensity (gMFI) of markers for AM identity, CD64, Siglec-F, and CD11b, in AMs (defined as CD11c+Siglec-F+ cells).(G) BrdU was injected 24h before euthanization of the mice. Representative FACS plot (left) and the quantification (right) of the percentage of AMs expressing Ki-67 or having BrdU incorporated in lungs from CbfbΔLysm or wild-type BM chimeric mice.RM one-way ANOVA with the Geisser-Greenhouse correction and multiple comparisons (C) and unpaired Student’s t test with Welch’s correction (D-G) were utilized for statistical evaluation. Data were shown as mean ± SD ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. Data in (B) were displayed in dots with diameter representing the percent of cells in the cluster (as shown in Figure 1B) that expressed the gene (or list of genes) indicated, with the depth of color indicating the average expression level. See also Figures S1C–S1E.
Fig 2: AMs from the aged mice possess senescence-like phenotypes(A) Module scores of cellular-senescence-related genes were calculated for the AMs.40 Violin Plot comparing the scores between AMs from young and aged mice (left), and the scores among the clusters as shown in Figure 3B (right).(B) Violin plots displaying selected senescence markers with gene names and protein names shown on the top respectively. (C and D) AMs were isolated from young and aged mice, followed by in vitro culture with different concentrations of GM-CSF (each dot represents one biological replicate).(C) Bar graph displaying the percentage of Ki-67 expressing AMs on in vitro stimulation with indicated concentration of GM-CSF.(D) Normalized percentage of Ki-67 expressing cells with that on saturated concentration of GM-CSF (10ng/mL) of AMs from young or aged mice.(E) Quantification of relative expression of selected markers for cellular senescence evaluated by qPCR. Each dot represents one biological individual.(F) Geometric mean of Fluorescence Intensity (gMFI) of SPiDER-βGal staining in AMs from lung homogenate of young (n = 4) or aged mice (n = 4).(G) Quantification of CBFβ in BAL cells from young (n = 3) and aged (n = 3) mice with western blot. The protein level was normalized to the total protein stain. Each dot represents one biological individual.(H) qPCR was performed with the cells in BAL from CbfbΔLysm or control mice. Data combined from 2 independent experiments. Each dot represents one biological replicate. Relative expression of selected markers for cellular senescence was displayed.two-Way ANOVA with multiple comparisons (C, D, and G, right) and unpaired Student’s t test with Welch’s correction (E, F, H and G, left) were utilized for statistical evaluation. Data were shown as mean ± SD ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001. The bar in (A) represents the median of module scores calculated with cellular-senescence-associated genes.40 See also Figures S3 and S4.
Supplier Page from BioLegend for Recombinant Mouse GM-CSF (carrier-free)