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. Author manuscript; available in PMC: 2022 Oct 14.
Published in final edited form as: Oncogene. 2022 Apr 14;41(20):2873–2884. doi: 10.1038/s41388-022-02310-0

Figure 1. The CRISPR/Cas9-mediated knockout of ID2 in Ewing sarcoma cell lines impairs growth in vitro and in vivo.

Figure 1.

(A) Immunoblot showing ID2 expression level after CRISPR/Cas9-mediated gene knockout in EW8 and TC71 cell lines. Results for two independent clones are shown for each cell line. (B-C) Colony formation assay with the ID2-KO and parental cell lines. Error bars represent the mean ± SD of three technical replicates. (D) Immunoblot showing re-expression of ID2 in the ID2-KO cell lines. (E-F) Colony formation assay with the parental cells (ID2-WT), ID2-KO cells, and the ID2-KO-Rescue cells. Error bars represent the mean ± SD of three technical replicates. (G) TC71 cells and two TC71-ID2-KO clones were implanted subcutaneously in NCr mice. Tumor size was quantified every 2–3 days using caliper measurements. Growth curves for each mouse cohort are shown until mice were removed from that cohort due to tumor size. (H) Kaplan-Meier survival curves for the different mouse cohorts. Mice were sacrificed when tumor reached >2 cm in any dimension. Log-rank (Mantel-Cox) test was used to calculate P-values comparing the survival curves for the mice with knockout cell lines compared to mice with the parental cell line. (I) Immunoblot showing the doxycycline-inducible, shRNA-mediated knockdown of ID2 in TC71 cells. (J) Colony formation assay with the TC71-shID2 cells in presence and absence of doxycycline. ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001.