Fig. 6: Altered estates in inflamed adipocytes are softened upon PKP2 restoration.

a A comprehensive pipeline was settled to sequentially validate altered cell cycle and enhanced senescence in MA under either defective PKP2 (si-PKP2) or enduring sustained exposure to MCM, also characterized by impaired PKP2 gene expression. In the latter, we used plasmids to restore PKP2 levels (MCM + OE_PKP2). Bar plots show the change for a set of genes previously identified as related to impaired PKP2, including those (i) directly or (ii) inversely related to senescence, and (iii) biomarkers downstream E2F signaling, also bound to cell cycle, in b si-PKP2 and c MCM-treated adipocytes. d SA-β-gal staining in MA under different conditions. Pictures show three representative images sorted out from four biological replicates in which measures of absorbance (546 nm) allowed the quantitative assessment plotted at the bottom right corner. e By using a BD Accuri C6 flow cytometer, the amounts of SA-β-gal positive adipocytes were assessed. Representative flow cytometric curves are provided for each treatment. The gray line shows cell distribution in unmarked control cells. The gating strategy is exemplified in supplementary Fig. S6c, d. The bean plot on the right indicates the amounts of SA-β-gal positive adipocytes for each condition (n = 4 biological replicates/condition). f Western blot analysis for PKP2, Cyclin D1 and PAPPA in non-targeting controls (NTC) and si-PKP2-treated MA. The bar plots below indicate β-actin-normalized relative amounts of each protein. g The box plot (median values plus 25^th and 75^th percentiles, and whiskers bound to maximum and minimum values in each group) shows concentrations of hydrogen peroxide (H₂O₂) measured in conditioned media supernatants collected from cell cultures of either NTC or si-PKP2 adipocytes (left boxes), or MCM-inflamed adipose cells (right boxes) containing a plasmid with an empty cassette (reference) or coding for PKP2 (n = 8 biological replicates/condition). h Representative 20x immunofluorescent images (the scale bars denote 100 μm length) showing cell cycle driver Cyclin D1 signal in adipocyte cultures. The bean plot at the right-hand shows the Cyclin D1 signal after correcting for blue fluorescent stain DAPI in four biological replicates/conditions. Below, i the percentage of nuclei showing Protein Kinase C alpha (PKCα) immunofluorescence staining in adipocyte cultures following the treatments depicted above. Representative 20x immunofluorescent images are provided in Fig. S6e (scale bars of 100 μm), and an example of image handling and data collection is shown in Fig. S6f. j Gene expression patterns and k Western blot analysis of PKP2, PAPPA, and Cyclin D1 in cultures of adipocytes maintained undisturbed (Ctrl) and inflamed (MCM) adipocytes in which we used plasmids to restore PKP2 levels (MCM + OE_PKP2). Ctrl and MCM groups were treated with the negative empty control vector for pReceiver-M90 and transfection reagent, as per proper control conditions. Bar plots show results assessed in four biological replicates/conditions, and the mean ± SEM Statistical significance was assessed by two-tailed Fisher’s exact t-test. *p < 0.05; **p < 0.01. Source data are provided as a Source data file.