Extended Data Fig. 1: Improvement of proliferation fitness of progeroid fibroblasts following FoxM1-dNdK short-term induction (related to Fig. 1).
(a) RT-qPCR analysis of endogenous FoxM1 transcript levels (2-ΔΔCt) following 2- and 4-days induction with doxycycline (dox; 2d, 4d). (b) RT-qPCR analysis of FOXM1-dNdK mRNA levels (2-ΔCt) in LAKI-FoxM1 fibroblasts. (c-f) RT-qPCR analysis of relative CcnB1 (c), Plk1 (d), p21/Cdkn1a (e) and p16/Cdkn2a (f) mRNA levels (2-ΔΔCt). (g) Western blot analysis of progerin/LMNA/C protein levels. Vinculin was used as loading control. (h) Quantification of progerin levels from western blot analysis. (i) Percentage of cells staining positive for the Annexin V-FITC apoptosis marker. (j) Western blot analysis of total FOXM1 protein levels in LAKI MAFs treated with dox for 2 and 4 consecutive days. (k) Quantification of total FOXM1 protein levels. Tubulin was used as loading control and levels were normalized to untreated cells (0d). (l-q) Immunofluorescence analysis of cellular aging phenotypes in LAKI MAFs following dox-treatment. (l) Percentage of proliferative cells (Ki67 + ). (m) Percentage of cells with DNA damage (γH2AX + ). (n) Percentage of cells staining positive for SA-β-gal activity assay. (o) Quantification of the fluorescence intensity levels of H3K9me3 and H4K20me3 epigenetic marks. (p) Percentage of cells with nuclear blebbing. (q) Quantification of the nuclear area. Error bars represent s.d. “n” in all graphs refer to the number of independent experiments, except for (o, q) where n = number of cells. Tbp and Gapdh were used as reference genes and expression levels were normalized to untreated LAKI-FoxM1 fibroblasts (0d). Statistics were performed by ordinary one-way ANOVA with Tukey’s multiple comparison correction (a-f, h, i, k), two-sided Fisher’s exact (l-n, p) and two-sided Kruskal-Wallis with Dunn’s multiple comparison correction (o, q) statistical tests for the indicated comparisons.
