Fig 2: Summary diagram. Progerin can be produced by genetic mutation (HGPS) or the normal aging process. The thickness of the blue arrow indicates the intensity of progerin expression. Human WRN (hWRN) binds to progerin and inhibits progerin expression under normal conditions. Under WRN-deficient conditions, such as in WRN, progerin can accumulate easily and promote cellular senescence. SLC-D011 binds to progerin and eliminates its expression. Thus, SLC-D011 can be used to treat WRN and normal aging as well as HGPS. The diagram was created with BioRender.com.

Fig 3: Gene ontology analysis of HGPS and WRN cells. (A) Heatmap of gene expression profiles in normal fibroblasts (young; N9; unaffected 9-year old subject, and aged; N81; unaffected 81-year-old subject) and prematurely aged cells (HGPS and WRN) at passage 10. Microarray was performed using Human Gene 1.0 ST arrays (Affymetrix). Yellow boxes indicate gene sets downregulated in both WRN and HGPS cells compared to normal fibroblasts (N9). Blue boxes indicate gene sets upregulated in both WRN and HGPS cells compared to N9 fibroblasts. White boxes indicate gene sets common to N81 fibroblasts as well as WRN and HGPS cells compared to N9 fibroblasts. (B) Analysis of differentially expressed genes (DEGs) showing a more than twofold difference from N9 fibroblasts. A total of 285 genes (core gene set) were commonly altered in progeroid cells (WRN and HGPS) and N81 fibroblasts compared to N9 fibroblasts. (C) Downregulated genes in the core gene set are associated with the cell cycle, chromosome separation, DNA replication, and DNA repair in HGPS, WRN, and N81 fibroblasts. Gene clustering was performed using the gene interaction mapping tool in cBioPortal (http://www.cbioportal.org). (D) DEG analysis (at least twofold) of HGPS and WRN cells compared to N9 fibroblasts. A total of 881 genes were altered in both HGPS and WRN cells. (E) Nine common elements overlapped between HGPS and WRN, including cell cycle regulation and chromosome separation. (F) Compared to normal fibroblasts (at passage 10), the expression of BRCA1, cyclin B1, cdc25c, rad51, H3K9me3, and p16INK4A in HGPS and WRN cells (at passage 10) was reduced. Western blots were cropped from different parts of the same samples and analyzed by film-based imaging systems (n = 3 independent experiments; two-tailed Student’s t-test).

Fig 4: LAP2 depletion exacerbates the progerin-induced proliferation defect whereas specific overexpression of LAP2a rescues it.(A) Growth curve of normal (ctrl shRNA) and LAP2-depleted (shLAP2) primary fibroblasts expressing progerin or lamin A. Dotted lines indicate SEM (n = 3). Inset: growth rate after 8 days, error bras indicate SEM (*p < 0.05, Student's t-test). (B) Western blot of primary and TERT+ cells expressing v5-tagged lamin A (v5-LA) or progerin (v5-PG) with or without LAP2 silencing by shRNA (shLAP2). V5-tag, lamin A, lamin C, progerin (PG), LAP2a, LAP2ß and GAPDH are indicated. (C) Growth curve of fibroblasts expressing control vector or progerin in the presence or absence of doxycycline-inducible LAP2a. LAP2a expression was induced by addition of 0.25 µg/ml doxycycline. Dotted lines indicate SEM (n = 3). Inset: growth rate after 5 days, error bras indicate SEM (**p < 0.01, Student's t-test). (D) Western blot of primary fibroblasts carrying doxycycline-inducible v5-LAP2a, expressing control vector or progerin. LAP2a expression was induced by addition of 0.25 µg/ml doxycycline. LAP2a, progerin and actin are indicated. (E) Western blot showing doxycycline-dependent induction of v5-LAP2a in TERT+HGPS cells. (F) Box plot of H3K27me3 levels in human fibroblasts expressing progerin in the presence (red) or absence (blue) of ectopically expressed LAP2a (Student's t-test, p < 0.05, n > 7500 cell analyzed, whiskers represent 10–90 percentile. (G) Scatter plot analysis of H3K27me3 levels in TERT+HGPS cells in the presence (red) or absence (blue) of ectopically expressed LAP2a showing increased levels of H3K27me3 upon LAP2a induction (Student's t-test, p < 0.01, n > 4000 cell analyzed. Inset: box plot of the same data, whiskers represent 10–90 percentile, ***p < 0.001). (H) Growth curve of control (ctrl) or progerin expressing fibroblasts (+progerin) in the presence or absence of doxycycline-inducible v5-LAP2ß. Dotted lines indicate SEM (n = 3). Inset: growth rate after 6 days, error bars indicate SEM (**p < 0.01, Student's t-test). (I) Western blot of fibroblasts transduced with doxycycline-inducible LAP2ß and expressing control vector or progerin. LAP2ß, progerin and actin are shown.DOI: http://dx.doi.org/10.7554/eLife.07759.013

Fig 5: miR-376a-3p and miR-376b-3p expression in human fibroblasts and LmnaG609G/G609G mouse model(A) hsa-miR-376a-3p and hsa-miR-376b-3p sequences; these two miRNAs belong to the miR-376 family and share 18 common nucleotides including the seed sequence (in blue); the last nucleotides at the 3' end are different (in red).(B) Expression of hsa-miR-376a-3p and hsa-miR-376b-3p quantified by RT-qPCR in HGPS fibroblasts (n = 4) as compared with the control fibroblasts (n = 4, unpaired t-test).(C and D) Expression of mmu-miR-376a-3p and mmu-miR-376b-3p quantified by RT-qPCR in LmnaG609G/G609G mice (n = 8) as compared with wild-type mice (n = 5) in (C) thoracic aorta and (D) aortic arch (unpaired t-test).(E) Expression (Cq) of mmu-miR-376a-3p and mmu-miR-376b-3p quantified by RT-qPCR in cultured VSMC from two aorta of LmnaG609G/G609G mice (n = 6) as compared with wild-type mice (n = 4) (ANOVA test). Data are represented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).