Chemical screen identifies a geroprotective role of quercetin in premature aging

Geng, Lingling; Liu, Zunpeng; Zhang, Weiqi; Li, Wei; Wu, Zeming; Wang, Wei; Ren, Ruotong; Su, Yao; Wang, Peichang; Sun, Liang; Ju, Zhenyu; Chan, Piu; Song, Moshi; Qu, Jing; Liu, Guang-Hui

Cell culture and differentiation WRN −/− hMSCs and LMNA G608G/+ hMSCs were obtained via the directed differentiation of WRN −/− hESCs and LMNA G608G/+ hESCs that we previously established Zhang et al., 2015) . hESCs were maintained on mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder in hESC culture medium containing 80% DMEM/F12 (Gibco), 20% Knockout Serum Replacement (Gibco), 10 ng/mL FGF2 (Joint Protein Central), 0.1 mmol/L non-essential amino acids (NEAA, Gibco), 2 mmol/L GlutaMAX (Gibco) and 55 μmol/L β-mercaptoethanol (Invitrogen). hESCs used in this study were also cultured on Matrigel (BD Biosciences) with mTeSR medium (STEMCELL Technologies). The human MSCs were cultured in hMSC culture medium containing 90% α-MEM + Glutamax (Gibco), 10% fetal bovine serum (FBS, Gemcell, Lot b Figure 9 . 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 Quetreated HGPS hMSCs (passage 10). Scale bar, 25 μm. Ki67positive 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.