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. 2020 Oct 19;39(23):e104500. doi: 10.15252/embj.2020104500

Figure 1. POT1‐loss triggers rapid telomere elongation and a DNA damage response throughout the cell cycle.

Figure 1

  1. Schematic drawing of the POT1 gene structure and two derived polypeptides. Exon 6 that has been chosen for gene editing is encircled.
  2. Schematic drawing of the repair template used for CRISPR/Cas9-mediated gene editing in HEK293E cells. Positions of primers for genotyping PCR are marked with arrows.
  3. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4‐OHT. Cl75—parental cell line. gRNA1—POT1 knockout in the population by transient transfection with pSpCas9(BB)‐2A-puro containing gRNA1 sequence. EV—corresponding empty vector.
  4. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0—time point 0, no 4OHT.
  5. Growth curve for POT1 WT and POT1 knockout cells. Population doublings (PDL) are represented as mean ± SD (three biological replicates).
  6. Quantification for TIFs in EdU‐positive and EdU‐negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two‐way ANOVA. P‐values are indicated on the graph.
Data information: 4‐OHT—4‐hydroxytamoxifen, NT—non‐treated cells.Source data are available online for this figure.