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. 2020 Oct 29;11:5469. doi: 10.1038/s41467-020-19205-x

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a CRISPR ‘knock-in’ strategies used to generate the conditional Zbtb11 KO allele (Zbtb11^lox) (left), and to insert the ERt2-Cre transgene in the Rosa26 locus (right). The loxP sites (lox) flanking Zbtb11 exon 3 include ectopic restriction sites (Supplementary Fig. 2a). Red arrowheads mark qPCR primers used to measure the deletion efficiency once ERt2-Cre is activated—recombination between the two loxP sites leads to excision of the left primer binding site along with exon 3, so PCR products are no longer generated. b Time-course experiment describing the dynamics of Zbtb11 inactivation in our inducible Zbtb11 KO ESC line (Zbtb11^lox/lox Rosa26^ERt2-Cre). Upper panel—decay of functional Zbtb11 alleles (red circles, n = 6 independent experiments, except 24 h timepoint for which n = 3), Zbtb11 transcripts (grey diamonds, n = 3) and protein (blue triangles, n = 3) in 4OHT-treated cells relative to their EtOH-treated control cells following KO induction. Lower panel—growth curves, with p values (two-tailed paired t test) above each timepoint. Source data are provided as a Source Data file. c Propidium iodide stain for cell cycle analysis of control (Zbtb11^lox/lox EtOH) and Zbtb11 KO (Zbtb11^lox/lox 4OHT) cells. 4OHT-treated Zbtb11^wt/wt cells were included to control for potential non-specific effects of 4OHT. Top two panels—representative flow cytometry histograms showing the distribution of DNA content in the sampled cell populations. G1, S or G2/M phases of the cell cycle are indicated on the horizontal axis. Bottom panel—mean and SD of the fractions of 4OHT-treated cells present in each cell cycle phase (normalised to EtOH-treated controls, n = 2). Source data are provided as a Source Data file. d Bright field microscopy images of 144 h Zbtb11 KO and control cells, illustrating the collapse of the culture 6 days after inducing Zbtb11 KO. e Quantification of cell death after Zbtb11 KO induction with FITC-coupled Annexin V. The fraction of Annexin V-positive cells is shown for n = 3 (48 h), n = 5 (72, 96 and 120 h) independent experiments (Supplementary Figs. 2d,9). Source data are provided as a Source Data file.