Fig 1: The proinflammatory cytokines were elevated in ALI patients. The concentrations of cytokines including IL-1β (A), IL-6 (B), IL-15 (C), TNF-α (D), IL-4 (E), and IL-13 (F) were measured using ELISA kits in serum samples obtained from 24 NSCLC patients (Control) under T0 stage and 24 ALI patients. ***P < 0.001.
Fig 2: Hypoxia-treated epithelial cells with senescence-associated phenotypes promote stromal cell proliferation through a HIF-1α-dependent SASP-like secretory program. (A-D) CCK-8 assays showing the proliferative responses of stromal cells after stimulation with conditioned media (CM) derived from prostate epithelial cells under different treatment conditions. (A) WPMY-1 cells treated with CM from BPH-1 cells in the NC, Hypoxia, and Hypoxia + siHIF1A groups. (B) WPMY-1 cells treated with CM from RWPE-1 cells in the NC, Hypoxia, and Hypoxia + siHIF1A groups. (C) PrSCs treated with CM from RWPE-1 cells in the NC, Hypoxia, and Hypoxia + siHIF1A groups. (D) PrSCs treated with CM from BPH-1 cells in the NC, Hypoxia, and Hypoxia + siHIF1A groups. Hypoxia-CM significantly promoted stromal cell proliferation, whereas HIF1A knockdown in epithelial cells attenuated this effect. (E-H) EdU assays showing DNA synthesis in stromal cells after CM stimulation. (E) WPMY-1 cells treated with BPH-1-derived CM. (F) WPMY-1 cells treated with RWPE-1-derived CM. (G) PrSCs treated with BPH-1-derived CM. (H) PrSCs treated with RWPE-1-derived CM. Scale bar = 10 μm. Hypoxia-CM increased the proportion of EdU-positive cells, and this effect was partially reversed by HIF1A knockdown. (I, L) Representative flow cytometric plots showing cell-cycle distribution of stromal cells after CM stimulation. (I) WPMY-1 cells treated with BPH-1- or RWPE-1-derived CM. (L) PrSCs treated with BPH-1- or RWPE-1-derived CM. (J, K, M, N) Quantification of the S-phase fraction in stromal cells after CM stimulation. (J) WPMY-1 cells treated with BPH-1-derived CM. (K) WPMY-1 cells treated with RWPE-1-derived CM. (M) PrSCs treated with BPH-1-derived CM. (N) PrSCs treated with RWPE-1-derived CM. Hypoxia-CM increased the proportion of stromal cells in S phase, whereas Hypoxia + siHIF1A-CM partially reversed this effect. (O-P) Detection of SASP-associated factors in epithelial CM. (O) Levels of IL-1α, IL-6, IL-8, IL-15, and CXCL12 in BPH-1-derived CM. (P) Levels of IL-1α, IL-6, IL-8, IL-15, and CXCL12 in RWPE-1-derived CM. Hypoxia increased the secretion of multiple SASP-associated cytokines and chemokines, and these increases were reduced after HIF1A knockdown. All data are presented as the mean ± standard deviation, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001.
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