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. 2021 Nov 12;13(22):5668. doi: 10.3390/cancers13225668

Figure 4.

Figure 4

FEZF1 knockdown impairs Ewing sarcoma cell proliferation. (A) mRNA expression levels of FEZF1, FEZF1-AS1 and EWSR1-FLI1 in three Ewing sarcoma cell lines expressing a doxycycline-inducible shRNA directed against FEZF1 mRNA. Cells were cultured for 72 h in the absence or presence of doxycycline (1 µg/mL), and mRNA expression levels were quantified by RT-qPCR (mean ± SD). (B) Western-blot analysis of FEZF1 and EWSR1-FLI1 in the same cell lines, confirming the downregulation of FEZF1. EWSR1-FLI1 protein levels were not affected by FEZF1 knockdown. (C) Cell colony formation assays show a reduction in the number of cell colonies formed upon FEZF1 knockdown. Scale bar: 10 mm. (D) The number of cell duplications accumulated over 30 days in which cells were cultured in the absence or presence of doxycycline. (E) Cells were cultured in the absence or presence of doxycycline (1 µg/mL), and DNA synthesis was analyzed by BrdU incorporation assay. FEZF1 knock-down significantly reduced DNA synthesis in the three Ewing sarcoma cell lines analyzed. For comparative purposes, the effect of EWSR1-FLI1 knock-down in A673/TR/shEF cells is also shown (mean ± SD of one experiment out of four with equivalent results; * p < 0.05, *** p < 0.001; Student’s t-test).