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. 2023 Jun 23;26(7):107197. doi: 10.1016/j.isci.2023.107197

Figure 3.

Figure 3

AMs from the aged mice display reduced self-renewal ability

(A) Schematic display of library preparation for scRNA-seq. AMs (CD11c+ Siglec-F+) were FACS-sorted from lung homogenate combined from young (∼2-month-old, n = 3) or aged (∼24-month-old, n = 3) female mice.

(B) UMAP displaying overall clustering of 10,531 AMs that passed quality control (upper left), split view with regard to their hosts (lower), and bar graph demonstrating quantification of cell number in each cluster (upper right).

(C) Cells were denoted to indicated phase in the cell cycle based on PCA analysis using genes sets of S phase and G2M phase, as displayed in UMAP (top) and quantification of cells in each phase (bottom).

(D) UMAP showing the trajectory analysis using Monocle3 with the proliferating cells as “stem cells”.

(E) Violin plot displaying Mki67 expression in AMs in young or aged mice from scRNA-seq. Quantification of percentage of cells expressing Mki67 was shown on the top.

(F and G) Flow cytometry of macrophages in the lung of young (n = 3) and aged mice (n = 3). F. Percentage of AMs expressing Ki-67 from the lung of young and aged mice shown by representative FACS plot (left) and quantification (right). G. Quantification of percentage and absolute number of AMs (left) and IMs (right).

(H) Colony formation assay using AMs from young (n = 3) and aged mice (n = 3). Demonstrated by representative pictures of the colony (left) and quantification of colony unit number (right).

(I) Bar graph showing selected results of pathway analysis comparing AMs from young and aged mice using GSEA.

(J) Bar graph showing selected results of pathway analysis comparing AMs from young and aged mice in published microarray data (GSE84901 using GSEA).

(K) Bar graph showing selected results of pathway analysis comparing AMs from young and aged mice in published bulk RNA sequencing data (GSE134397 using GSEA).

Unpaired Student’s t test with Welch’s correction (F, G and H) or GSEA (I, J and K) were utilized for statistical evaluation. Data were shown as mean ± SD ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001. See also Figure S2.