Journal: bioRxiv

Article Title: High-altitude hypoxia exposure inhibits erythrophagocytosis by inducing macrophage ferroptosis in the spleen

doi: 10.1101/2023.03.23.533972

Figure Lengend Snippet: HH exposure causes a decrease in the number of macrophages in spleen. The C57BL/6 mice were treated with NN and HH for 7 or 14 days, and the spleen and blood were collected for subsequent detection. ( A ) Visualization of spleen cell clusters using uniform manifold approximation and projection (UMAP). Each color represents a cluster of specific cells of the spleen, which was identified by a defined gene expression profile. ( B ) The proportions of different macrophage clusters derived from either NN-or HH-treated mouse spleens were analysed. ( C ) Individual macrophages derived from either NN-or HH-treated mouse spleens are denoted. ( D ) Calcein/PI double staining was analysed by flow cytometry. ( E and F ) Cell death proportions in total splenic cells are given as bar graphs. ( G and J ) Western blot detecting CD16 and CD206 protein expression in the spleen. ( H-I and K-L ) Statistical analysis of CD16 and CD206 expression in H and K. ( M ) F4/80 staining with CD11b, CD86 or CD206 in splenic cells was analysed by flow cytometry. ( N ) qPCR analysis of CCL2 and CCL7 expression in the spleen. ( O ) qPCR analysis of Csf1 and Csf2 expression in the spleen. ( P ) Flow cytometry detection of CD111b and Ly6C double-stained cells in the BM and spleen. CD11b hi Ly6C hi cell proportions in the BM ( Q-R ) and spleen ( S-T ) are given as bar graphs. ( U ) HO-1 and F4/80 expression in the spleen after HH exposure for 7 and 14 days. Data were expressed as the means ± SEM (n = 3 per group); * P < 0.05, ** P < 0.01, *** P < 0.001 versus the NN group or the indicated group.

Article Snippet: The membranes were incubated with the following antibodies: HO-1 (1:2000, Abcam, ab13243, USA); Ft-L (1:1000, Abcam, ab69090, USA); Ft-H (1:1000, Novus, NBP1-31944, USA); NCOA4 (1:1000, Santa Cruz, sc-373739, USA); TfR (1:1000, Thermo Fisher, 13-6800, USA); Fpn (1:1000, Novus, NBP1-21502, USA); ACSL4 (1:1000, Santa Cruz, sc-271800, USA); xCT (1:1000, Proteintech, 26864-1-AP, USA); Gpx4 (1:1000, Abcam, ab125066, USA); CD206 (1:1000, RD, AF2535, USA); CD16 (1:2000, RD, AF1960, USA); and β-actin (1:1000, Sigma‒Aldrich, A5316, USA).

Techniques: Expressing, Derivative Assay, Double Staining, Flow Cytometry, Western Blot, Staining