Fig 2: Que alleviated senescence in HGPS hMSCs with a synergistic effect with VC. (A) Confirmation of the heterozygous mutation of LMNA by DNA sequencing. (B) RT-qPCR analysis of progerin mRNA expression in vehicle-, Que-, VC- and VC plus Que-treated HGPS hMSCs (passage 10). Data are shown as mean ± SEM (n = 3). *P < 0.05 vs. Ctrl; **P < 0.01 vs. Ctrl. (C) Immunostaining of progerin in vehicle-, Que-, VC- and VC plus Que-treated HGPS hMSCs (passage 10), Scale bar, 25 μm. Mean fluorescence intensity on the right is shown as mean ± SEM (cell number ≥ 300). ***P < 0.001 vs. Ctrl. (D) Accumulative growth curve showing the population doubling of HGPS hMSCs upon the treatment of vehicle, Que, VC and VC plus Que (n = 3). (E) Analysis of SA-β-Gal activity in vehicle-, Que-, VC- and VC plus Que-treated HGPS hMSCs. Left, representative images of SA-β-Gal staining (passage 10); right, frequency of SA-β-Gal-positive cells. Data are shown as mean ± SEM (n = 3). **P < 0.01 vs. Ctrl; ***P < 0.001 vs. Ctrl. Scale bar, 100 μm. (F) Clonal expansion ability of vehicle-, Que-, VC- and VC plus Que-treated HGPS hMSCs (passage 8) stained by crystal violet. Quantitative data are shown as mean ± SEM (n = 3). *P < 0.05 vs. Ctrl; ***P < 0.001 vs. Ctrl. Scale bar, 100 μm. (G) Immunostaining of Ki67 in vehicle- and Que-treated HGPS hMSCs (passage 10). Scale bar, 25 μm. Ki67-positive cells are shown as mean ± SEM (cell number ≥ 300). *P < 0.05 vs. Ctrl. (H) Quantitative PCR analysis of telomere length in vehicle- and Que-treated HGPS hMSCs (passage 10). Data are shown as mean ± SEM (n = 3). *P < 0.05 vs. Ctrl. (I) Immunostaining of Lamin A/C in vehicle- and Que-treated HGPS hMSCs (passage 10). Quantifications of abnormal nuclei of HGPS hMSCs on the right are shown as mean ± SEM (n ≥ 300). **P < 0.01 vs. Ctrl. Scale bar, 10 μm

Fig 3: Nuclear morphology and immunohistological staining of lamins in skin tissue and pre-adipocytes.(a). Immunohistological staining of lamins in abdominal skin biopsies from the female patient. (b). Abnormal features of the nuclear envelope in 3T3-L1 pre-a dipocytes transfected with wild-type LMNA or the R133L mutant LMNA (400X, arrows point to different nuclei abnormalities).

Fig 4: Stable expression of human LMNA R133L impairs adipocyte differentiation and insulin pathway signalling.(a). Expression of human LMNA wild-type or LMNA R133L as detected by RT-PCR in three clonal lines of 3T3-L1 cells transfected with control lentivirus, or lentivirus encoding Flag-tagged human LMNA wild-type or LMNA R133L. (b). Western blotting of lamin A/C and Flag-tagged proteins in three clonal lines of 3T3-L1 cells. (c). Red-Oil staining of three clonal lines of 3T3-L1 cells after 10 days of differentiation (200 X). (d). Expressions of adipogenic or lipogenic genes in each of three clonal lines of 3T3-L1 cells transfected with control lentivirus, or lentivirus encoding Flag-tagged human LMNA wild-type or LMNA R133L induced over 4 days and 10 days differentiation as revealed by qPCR using gene-specific primers (n = 3). Data are presented as means ± SDs. *P < 0.05 vs. Control; **P < 0.01 vs. Control. #P < 0.05 vs. LMNA wild-type; ##P < 0.01 vs. LMNA wild-type. (e). PI3K/Akt signalling pathway activity upon insulin treatment of three clonal lines of 3T3-L1 cells after 4 days of differentiation.

Fig 5: LMNA gene analysis in the pedigree.(a). Genetic analysis of the LMNA gene of the patient and her pedigree. (b). The search for the LMNA R133L mutation in the patient’s relatives (arrow: proband). (c). Sequence conservation of R133 in vertebrate LMNA data in the Swiss-Prot database (with accession numbers) and alignments around the conserved R133 residue (red).