Figure 3. The isoprenylcysteine carboxylmethyltransferase inhibitor C75 disrupts progerin-AKT interactions, increases AKT activation, and mislocalizes progerin from the nuclear membrane to the nucleoplasm.

(a) Western blot showing amounts of phosphorylated and total AKT (a.k.a protein kinase B) in whole-cell lysates of human wild-type (WT) and Hutchinson-Gilford progeria syndrome (HGPS) cells and mouse Zmpste24-deficient fibroblasts. (b, c) Left panels, western blots (WB) showing amounts of phosphorylated and total AKT in WT and HGPS cells (b) and Zmpste24-deficient (c) fibroblasts incubated with C75 for 20–30 days; β tubulin was the loading control. Right panels, quantification of the ratio of phosphorylated and total AKT from densitometry analyses of protein bands. Data are mean of two independent cell lines, each analyzed in duplicate. (d) Immunoprecipitation (IP) and WB analyses showing that C75 disrupts the association between AKT and progerin. The lysates were also used directly for WB with total AKT antibodies (input). (e) WB showing amounts of progerin in HGPS cells incubated with C75 for 20 days. (f) WB showing amounts of progerin remaining in HGPS cells incubated with vehicle and 5 μM C75 for 20 days and then with cycloheximide to stop protein synthesis. (g) Quantification of progerin amounts from the experiment in panel f and two others like it. (h, i) Left panels, WB showing amounts of progerin and prelamin A in nuclear membrane and nucleoplasm fractions of HGPS (h) and Zmpste24-deficient (i) fibroblasts, respectively, incubated with vehicle and 5 μM C75 for 20 days. Lamin B2 and nuclear P84 were loading controls for nuclear membrane and nucleoplasm fractions, respectively. Right panels, quantification of nucleoplasmic progerin/prelamin A from densitometry analyses. *p<0.05; **p<0.01; ***p<0.001.