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. 2023 Oct 12;18(11):2174–2189. doi: 10.1016/j.stemcr.2023.09.009

Figure 5.

Figure 5

Sall4 and Nanog tracing systems cannot explain the reprogramming blockage observed in the double heterozygous mutant cells

(A and B) Schematic representation of the targeting strategy to introduce a 2A-EGFP-ERT-CRE-ERT cassette into the Sall4 locus (A) or into the Nanog locus (B).

(C and D) PCR validations for targeted colonies demonstrate correct targeting band size for Sall4 (C) and for Nanog (D) using both 5′ and 3′ regions of the incorporation point. Black arrows depict correct targeting events. NC, negative control.

(E and F) Representative bright field, RFP, and GFP channel images for the Sall4 (E) or Nanog (F) tracing systems before and after Tam addition.

(G) Flow cytometry analysis of tdTomato-positive RL8 induced cells that were infected with dox-inducible OSKM vectors and exposed to dox with or without Tam for 6 days.

(H) Graph summarizes the percentages of tdTomato-positive cells of the Sall4 tracing system after 6 days of dox with or without Tam. Error bars indicate standard deviation between 7 independent experiments/replicates (n = 7). ∗∗∗∗p < 0.0001 using a two-tailed unpaired t test calculated by GraphPad Prism (8.3.0).

(I) Bright field and RFP channel images of tdTomato-positive cells from the Sall4 tracing system after 6 days of dox and Tam addition.

(J) Flow cytometry analysis of tdTomato-positive RL9 induced cells that were infected with dox-inducible OSKM vectors and exposed to dox with or without Tam for 6 days.

(K) Graph summarizes the percentages of tdTomato-positive cells of the Nanog tracing system after 6 days of dox with or without Tam. Error bars indicate standard deviation between 6 independent experiments/replicates (n = 6). ∗∗∗∗p < 0.0001 using a two-tailed unpaired t test calculated by GraphPad Prism (8.3.0).

(L) Bright field and RFP channel images of tdTomato-positive cells from the Nanog tracing system after 6 days of dox and Tam addition.

(M and N) Flow cytometry analysis of tdTomato and SALL4-2A-EGFP-positive cells (M) or NANOG-2A-EGFP-positive cells (N) after 13 days of OSKM induction in the presence of dox and Tam followed by 3 days of dox withdrawal. Representative flow cytometry plots are shown out of 7 or 6 independent reprogramming runs (n = 7 for Sall4 and n = 6 for Nanog tracing). See also Figure S5.