Figure 2. C75 inhibits isoprenylcysteine carboxylmethyltransferase (ICMT) activity and increases proliferation and reduces senescence of HGPS cells without affecting nuclear shape.

(a) Chemical structure of compound 75 (C75) and percent ICMT activity remaining after incubation with C75. (b) Western blot showing increased amounts of prelamin A in Hutchinson-Gilford progeria syndrome (HGPS) and wild-type (WT) cells incubated with C75 for 20 days; β tubulin was the loading control. (c) Left, immunofluorescence images showing prelamin A expression in HGPS cells incubated with 5 μM C75 for 20 days and 10 μM farnesyltransferase inhibitor (FTI) for 3 days; the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Right, quantification of prelamin A staining intensity (n = 2 cell lines and six individual images/cell line). (d) Left, representative nuclei of LAP2β-stained WT cells and HGPS cells incubated with vehicle (Ctrl), 5 μM C75 for 20 days, and 10 μM FTI for 3 days. Right, quantification of misshapen nuclei. Data are mean of ∼1000 nuclei and two independent experiments per cell line and condition. (e–h) Population doubling assays of late-passage HGPS cell lines (e, f), primary Zmpste24–/– mouse fibroblasts (g), and Zmpste24–/–Icmt–/– fibroblasts (h) incubated with vehicle (Ctrl) and C75. (i) Population doubling of WT cells incubated with C75. (j, k) Population doubling assays of a late-passage HGPS cell line incubated with vehicle (Ctrl), lonafarnib, C75, and both drugs. (l–n) Senescence-associated beta-galactosidase (β-gal) staining of WT, HGPS, and Zmpste24–/– fibroblasts incubated with vehicle (Ctrl) and C75 for 20 days. (o, p) Interleukin 6 (IL-6) (o) and CDKN2A (p) expression in cells from experiment in panel m. **p<0.01; ***p<0.001; ****p<0.0001; n.s., not significant.

Figure 2—figure supplement 1. C75 synthesis and absorption, distribution, metabolism, and excretion (ADME) test.

(a) Schematic of the C75 synthesis reactions. (b) Mass spectra of the final product. (c) Nuclear magnetic resonance spectra of C75. (d) Left, schematic of the apparent permeability assay through a layer of Caco-2 cells. Right, quantification of apical-to-basolateral (blue) and basolateral-to-apical (red) absorption rates across a Caco-2 cell layer.

Figure 2—figure supplement 2. C75 increases viability, proliferation, and metabolic rates of Hutchinson-Gilford progeria syndrome (HGPS) cells and reduces reactive oxygen species (ROS) levels and nuclei with high levels of DNA damage.

(a) Western blots showing levels of the RAS oncogene in total lysates (T) and in cytosolic (C) and membrane (M) fractions of late-passage HGPS cells and primary Zmpste24–/– mouse fibroblasts; β-actin was the loading control for cytosolic and total fractions. (b) Western blots showing electrophoretic mobility of the CAAX-protein HDJ2; a farnesyltransferase inhibitor (3-day incubation), but not C75 (20-day incubation), could partially reduce HDJ2 mobility. (c and d) Growth curves from presto blue-based cell viability assays of two HGPS cell lines. (e) Cell cycle analysis of HGPS cells. Data are mean of six technical replicates. (f) Seahorse analyses of basal and maximal respiration, and ATP production. Data are mean of two HGPS cell lines assayed in triplicate. (g) ROS levels in two HGPS cell lines incubated with C75 for 20 days. (h) Western blots showing levels of endoplasmic reticulum stress markers in lysates of two HGPS cell lines incubated with C75 for 20 days; tunicamycin was used as positive control. (i) Western blots showing levels of DNA damage markers in lysates of two HGPS cell lines incubated with C75 for 20 days. (j) Left, quantification of the frequency of γ-H₂AX-positive nuclei in wild-type cells and in HGPS cells lines incubated with 5 μM C75 for 20 days. Middle, quantification of nuclei with more than six γ-H2AX-foci HGPS cells incubated with 5 μM C75 for 20 days. Data are mean of two cell lines and triplicate analyses. Right, immunofluorescence images showing γ-H₂AX-positive nuclei (red); the nuclei were counterstained with DAPI (blue). *p<0.05; **p<0.01; ***p<0.001; n.s., not significant.