Fig. 1: Induction of senescence in Mdm2 mice by AAV8.TBG.Cre dosing leads to time sensitive inflammatory and regenerative macrophage responses.

Purple dashed lines are healthy control mean ± SEM. Data points represent mean of N = 3–5 independent animals per timepoint analysed. All error bars are ±SEM. All qPCR results were normalised to the PPIA housekeeper and expressed as fold change relative to healthy control mean expression. For histological staining quantification the % of the field of view (FOV) positively stained was assessed, with ≥10 FOVs analysed per animal. a Experimental schematic for senescence dose response study. Inset demonstrates Cre recombinase expression mediated loss of Mdm2 in hepatocytes as a result of injection of hepatotropic AAV8.TBG.Cre. Timepoints for tissue collection and analysis are indicated by days since injury induction (e.g. D3). Elements of this panel were produced using biorender.com. b, c Expression of the p21 senescence marker at D3, D7 and D14 following AAV8.TBG.Cre induction analysed by qPCR and histological staining. Results show a significant, time sensitive increase in senescent marker expression, with expression resolving to healthy control animal levels by D14. Ordinary One-Way ANOVA relative to healthy control mean with Dunnett’s Multiple Comparisons test. Scale bars 100 µm. d Representative histological micrographs and quantification of the pan macrophage marker F4/80 at D3, D7 and D14 post-induction, alongside healthy control. Scale bars 50 µm. Results demonstrate no overall increase in the density of macrophages as a result of senescence driven injury and are supported by limited fold changes in gene expression of the pan macrophage markers EMR-1 (F4/80) and CD68, as analysed by qPCR (e). f Time sensitive changes in gene expression of the inflammatory macrophage markers CD80 and CD86 as a result of senescence induction. g Representative immunofluorescent micrographs of the pro-regenerative macrophage marker CD206 at D3, D7 and D14 post-induction, alongside healthy control. Scale bars 20 µm (yellow inset) and 100 µm (white). White arrows indicate positively stained cells. Insets are digitally magnified 1.5x. h Quantification of CD206 staining, showing a time sensitive increase in staining. i Analysis of gene expression of the pro-regenerative macrophage markers CD163 and CD206.