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. 2019 Sep 25;11(10):1427. doi: 10.3390/cancers11101427

Figure 7.

Figure 7

FOXO1 induces growth inhibition in BL cell lines (A–E) BL cell lines were transduced with lentiviral vectors expressing constitutively active FOXO1(3A) ER (F1ER) vs. empty vector (EV) control. (A) Transduced cells were FACS sorted and lysed 2 days post induction with 100 nM 4-OHT. Expression levels of FOXO1, cleaved CASP3 and CDKN1B were analyzed by immunoblot. TUBB served as loading control. A representative of 3 independent experiments is shown. (B) Growth dynamics of transduced BL cell lines. The percentage of GFP+ cells was measured every 3 days using flow cytometry. First measurement was performed 4 days post transduction and the percentage of GFP+ cells was set as 100 %. Data are shown as mean ± SD (n ≥ 3). (C) Cell death analysis of BL cell lines. Transduced cells were FACS sorted, followed by AnnexinV-APC/PI staining 2- and 4-days post induction with 100 nM 4-OHT. After FACS measurement, Specific Apoptosis (SA) was calculated as SA (%) = 100 × (AE−AC)/(100−AC), where AE equals the percentage of apoptotic cells in the experimental group and AC equals the percentage of apoptotic cells in the control group. Data are shown as mean ± SD (n = 3). The data were analyzed by two-sided T-test. *, p < 0.05, **, p < 0.01. Representative dot-plot images are shown. (D) FOXO1 overexpression inhibits cell cycle progression. Transduced cells were FACS sorted, followed cell cycle analysis by PI staining 2- and 4-days post induction with 100 nM 4-OHT. Data are shown as mean percentage of cells in a cell cycle phase ± SD (n = 3). The data were analyzed by two-sided T-test. *, p < 0.05, **, p < 0.01. (E) qRT-PCR analysis of FOXO1 target gene TNSF10. Transduced cells were FACS sorted and RNA was isolated 2 days post induction with 100 nM 4-OHT. qRT-PCR data were quantified by the 2−ΔΔCT method. Data are shown as mean ± SD (n = 3). (F,G) BL cell lines were transduced with lentiviral vectors expressing wildtype FOXO1 (F1(WT)) vs. EV control. (F) Transduced cells were FACS sorted 4–5 days post transduction. Expression levels of FOXO1 were analyzed by immunoblot. TUBB served as loading control. A representative of 2 independent experiments is shown. (G) Growth dynamics of transduced BL cell lines. The percentage of GFP+ cells was measured every 3 days using flow cytometry. First measurement was performed 4–5 days post transduction and the percentage of GFP+ cells was set as 100 %. Data are shown as mean ± SD (n = 3).