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. 2020 Jun 7;39(26):4970–4982. doi: 10.1038/s41388-020-1333-1

Fig. 3. ELDA shows ZSCAN4 induction increases the frequency and size of tumorspheres and tumors.

Fig. 3

a Induction of ZSCAN4 in tet-ZSCAN4 Tu167 cells and b 012SCC cells by addition of doxycycline for 48 h (Dox+), prior to generation of tumorspheres, significantly increases the number and size of tumorspheres compared with untreated isogenic controls (Dox−) and to wild type (WT) treated or untreated with Dox. The statistical significance between the groups was determined using two away ANOVA with multiple Tukey’s post hoc comparisons. Asterisks indicate a significant difference from isogenic untreated and WT cells: **p < 0.01, ***p < 0.001. c Illustration of Extreme limiting dilution assay (ELDA) in vivo in NGS immunodeficient mice. tet-ZSCAN4 (Tu167) cells were treated (Dox+) or untreated (Dox−) with Dox for 48 h in culture. No Dox was given to the mice throughout the rest of the experiment. Cells were injected subcutaneously into the right and left flank of NOD/SCID gamma immunodeficient mice in multiple increasing dilutions: 100,000 cells, 10,000 cells, and 1000 cells per inoculation (n = 8), and allowed to form tumors for up to 85 days. d, e Tumor growth shown are mean ± SEM for each group (100,000 n = 6; 10,000 n = 8; 1000 n = 8).