Extended Data Fig. 1: Generation and characterization of APOE+/+ and APOE-/- hESCs.
From: Destabilizing heterochromatin by APOE mediates senescence
a, RT-qPCR showing the mRNA levels of APOE and CDKN1A (P21) in RS-hMPCs (P3), HGPS-hMPCs (P3), and WS-hMPCs (P3). GAPDH was used as the internal control. b, Western blot analysis of APOE in hMPCs (P4) transduced with lentiviruses expressing luciferase (Luc) or APOE. c, Schematic diagram of CRISPR-dCas9-mediated transcriptional activation of endogenous APOE. d, Western blot analysis of APOE in hMPCs transfected with non-targeting (negative control, sg-NC) or APOE-targeting (sg-APOE) sgRNAs using the CRISPR-dCas9 transcriptional activation system. e, Schematic diagram of APOE knockout strategy through CRISPR-Cas9-mediated non-homologous end joining (NHEJ). Sequencing results showed a 1-bp (T/A) insertion introduced by genome editing. f, Western blot analysis of the APOE protein level in APOE+/+ hESCs and APOE-/- hESCs. g, Immunofluorescence analysis of pluripotency markers OCT4, NANOG, and SOX2 in APOE+/+ hESC and APOE-/- hESC. Scale bars, 25 μm. Phase-contrast images were shown on the right. Scale bars, 250 μm. h, Immunofluorescence analysis of TuJ1, SMA, and FOXA2 to evaluate the differentiation potential of APOE+/+ hESC and APOE-/- hESC toward lineages of all three germ layers, that is, ectoderm, mesoderm, and endoderm, respectively. Scale bars, 25 μm. i, Cell cycle analysis of APOE+/+ hESCs and APOE-/- hESCs. j, Immunofluorescence analysis of Ki67 in APOE+/+ hESCs and APOE-/- hESCs. Scale bars, 25 μm. k, Detection of the DNA methylation status of the OCT4 promoter in APOE+/+ hESCs and APOE-/- hESCs. l, Karyotype analysis of APOE+/+ hESCs and APOE-/- hESCs. m, The copy number variation (CNV) analysis by whole genome sequencing of APOE+/+ hESCs and APOE-/- hESCs. a,i–j, A Two-tailed Student’s t-test was used for statistical analysis. Data are presented as the means ± s.e.m. a, n = 4 biological repeats. b,d,f,g, n = 3 biological repeats. b,d,f, For western blot analysis, β-Tubulin was used as the loading control.
