Fig. 7

TERF1 expression levels associated with telomere length and pluripotency of human embryonic stem cell (hESCs). a Scheme for hTERF1 knockout by CRISPR/Cas9. b Morphology of TERF1 ^+/- and wild-type (WT) hESCs (WA26) at passage 11. Scale bar = 100 μm. c Expression levels of pluripotency and telomerase genes in TERF1 ^+/- and WT hESCs by qPCR analysis. *P < 0.05, **P < 0.01, compared with WT controls. The data values of each gene are provided in Additional file 18: Table S10. d Western blot analysis of TERF1, SOX2, and OCT4/POU5F1 protein levels in TERF1 ^+/- and WT hESCs. β-actin served as a loading control. e Cell cycle analysis of TERF1 ^+/- and WT hESCs by flow cytometry. Data represent mean ± SD of three independent experiments. f Telomerase activity by TRAP assay of TERF1 ^+/- and WT hESCs at different passages. U2OS served as a negative control. g Telomere length measurement by southern blot analysis of TERF1 ^+/- and WT hESCs. h Quantification of southern blot results. Data represent mean ± SD of two independent experiments. i Immunofluorescence detection of γH2AX and telomere FISH of TERF1 ^+/- and WT hESCs. j, k Number of telomere-γH2AX colocalization foci per cell (j) and percentage of cells with the colocalization (k). l Relative telomere length of individual TERF1 ^+/- and WT hESCs by scT&R-seq. Right panel shows the mean telomere length by scT&R-seq. m Gene expression measured in single cells of TERF1 ^+/- and WT hESCs by scT&R-seq. Right panel shows the mean level of gene expression. n Linear regression analysis of telomere length and gene expression (TERF1 and NANOG) at single cell level