Fig. 3: Hypoxia in synergy with IL-1β triggering of IL-1RI mediate G2/M arrest and senescence of human primary PTECs. | Cell Death & Disease

Fig. 3: Hypoxia in synergy with IL-1β triggering of IL-1RI mediate G2/M arrest and senescence of human primary PTECs.

From: Human proximal tubular epithelial cell interleukin-1 receptor signalling triggers G2/M arrest and cellular senescence during hypoxic kidney injury

Fig. 3

A Fold changes (relative to Normoxia+Vehicle) in cell proliferation (MTT assay) for PTECs cultured for 72 h under normoxic (norm) or hypoxic (hypox) conditions in the absence (vehicle; veh) or presence of IL-1β. Bar graphs represent mean ± SEM. Symbols represent individual donor PTECs; n = 8. **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey’s multiple-comparison test. B Left panel: Representative cell cycle analysis for PTECs cultured for 72 h under normoxic (norm) or hypoxic (hypox) conditions in the absence (vehicle; veh) or presence of IL-1β. The percentage of cells in G2/M phase arrest for each histogram are presented. Representative gating strategy showing exclusion of debris/dead cells and aggregates/doublets prior to cell cycle analysis is outlined in Fig. S2C. Right panel: Cell cycle analysis (% of cells in each cell cycle stage) for PTECs under different culture conditions. Bar graphs represent mean ± SEM. Symbols represent individual donor PTECs; n = 9. *P < 0.05, **P < 0.01, one-way ANOVA with Tukey’s multiple-comparison test. C Left panel: Fold changes (relative to Normoxia+Vehicle) in senescence associated β-galactosidase (SA-β-gal) activity (measured as % SA-β-gal+ cells) for PTECs cultured for 72 h under normoxic (norm) or hypoxic (hypox) conditions in the absence (vehicle; veh) or presence of IL-1β. Bar graphs represent mean ± SEM. Symbols represent individual donor PTECs; n = 6. *P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test. Right panel: Representative SA-β-gal staining for PTECs cultured under normoxic (norm) or hypoxic (hypox) conditions in the absence (vehicle; veh) or presence of IL-1β. Scale bars represent 100 µm. D Left panel: Fold changes (relative to Normoxia+Vehicle+Isotype Ab) in SA-β-gal activity (measured as % SA-β-gal+ cells) for PTECs cultured for 72 h under normoxic conditions alone (Norm+Veh) or hypoxic conditions with IL-1β (Hypox+IL-1β) in the presence of isotype control antibody (Iso Ab) or neutralising IL-1RI Ab. Bar graphs represent mean ± SEM. Symbols represent individual donor PTECs; n = 7. *P < 0.05, ***P < 0.001, one-way ANOVA with Tukey’s multiple-comparison test. Right panel: Representative SA-β-gal staining; scale bars represent 100 µm. E Left panel: Fold changes (relative to Normoxia+Vehicle+DMSO) in SA-β-gal activity (measured as % SA-β-gal+ cells) for PTECs initially cultured for 72 h under normoxic conditions alone (Norm+Veh) or hypoxic conditions in the presence of IL-1β (Hypox+IL-1β), followed by additional 24 h treatment in fresh DM without (DMSO vehicle control) or with quercetin+dasatinib (Q + D). Bar graphs represent mean ± SEM. Symbols represent individual donor PTECs; n = 7. *P < 0.05, one-way ANOVA with Tukey’s multiple-comparison test. Right panel: Representative SA-β-gal staining; scale bars represent 100 µm.

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