Extended Data Fig. 5: No difference in canonical senescence pathways.
From: Neonatal imprinting of alveolar macrophages via neutrophil-derived 12-HETE
(a) Representative histograms and quantification of p53 MFI in PND3 AM from WT (n = 5) and Alox15−/− (n = 6) animals. (b) Expression of Trp53 in adult AM from WT and Alox15−/− mice (n = 6/group). (c-f) Representative histograms and quantification of p53 (c) (n = 5/group), MitoSox (d) (n = 3/group), p-p38 (e) (n = 3/group) and p-γH2AX (f) (n = 5/group) in BAL AM isolated from adult WT and Alox15−/− mice. (g-h) Heatmap and quantification of genes involved in antioxidant response (g) or interferon response (h) in BAL AM isolated from adult WT (n = 3) and Alox15−/− (n = 4) mice in bulk RNA-Seq dataset. (i) Expression of Ptgs/Ptges (WT n = 5/group, Alox15−/− n = 6/5/6/6/6/group) and Ptger genes by BAL AM from adult WT and Alox15−/− mice (n = 6/group). (j) Expression of Ptgs1 by AM, PM and BMDM from adult WT and Alox15−/− mice (n = 6/group). (k-l) PGE2 production by naïve resting (24 h) peritoneal macrophages (k) or bone marrow derived macrophages (l) from adult WT and Alox15−/− mice (n = 6/group). (m-n) BAL AM were isolated from adult (6–8 weeks) WT mice, and they were cultured or not with GM-CSF (20 ng/ml) for 3 days and treated or not with various concentrations of exogenous PGE2. (m) Representative micrographs of Ki67+ AM after 3 days of culture with GM-CSF and treated or not with PGE2 (1–10 µM). Bar = 50 µm. (n) AM growth was assessed after culture with GM-CSF and treatment with PGE2 (10 µM) (n = 3 biological replicates/group). (o) Heatmap and quantification of lipoxygenase pathway related genes in BAL AM from adult WT (n = 3) and Alox15−/− (n = 4). (p) Production of LTB4 and cysteinyl leukotrienes by resting adult WT and Alox15−/− BAL AM (24 h) (n = 3 biological replicates/group). Data are presented as mean ± s.e.m and are from one (e, g-h, o) or pooled from two (b, i-l) or three (n, p) independent experiments or representative of two experiments (a, c-f, m) independent experiments. Data were analysed using two-way ANOVA followed by Sidak’s multiple comparisons test (i-j, n).
