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

Figure 1.

Figure 1

FOXO1 knockdown negatively regulates proliferation of BL cell lines. (A,B) BL and cHL cell lines were transduced with lentiviral vectors expressing FOXO1 shRNA (F1sh) or FOXO shRNA1/3/6 (Fsh1/3/6) vs. scrambled (scr) control. (A) Knockdown efficiencies of F1sh and Fsh1/3/6 vs. scr control. Transduced cells were selected for 2 days using 4 µg/mL puromycin and FOXO1 expression was analyzed 5–6 days post transduction. Expression of TUBB served as loading control. A representative of 2–3 independent experiments is shown. (B) Growth dynamics of transduced BL and cHL cell lines. The percentage of RFP+ cells was measured every 3 days using flow cytometry starting from day 4 post transduction. The percentage of RFP+ cells at begin of measurements (day 0) was set as 100. Data are shown as mean ± SD (n ≥ 3). (C) BL cell line Namalwa was transduced with lentiviral vectors co-expressing Cas9 with sgRNAs targeting FOXO1 (sgF1.1, sgF1.2) or non-targeting (NT) control. For constructs expressing sgRNAs, RFP+/FOXO1− cell population was tracked (Figure S1C). For NT control, RFP+/FOXO1+ cell population was tracked, due to the apparent absence of RFP+/FOXO1− population in this group. The samples were measured every 3 days by flow cytometry. First measurement was performed 4 days post transduction and the percentage of the indicated cell population was set as 100. Data are shown as mean ± SD (n = 2). (D) FOXO1 knockdown inhibits cell cycle progression. BL cell lines expressing F1sh or scr vectors were sorted 4 days post transduction, followed by cell cycle analysis by PI staining. 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, ***, p < 0.001. (E) Cell death analysis of BL cell lines expressing F1sh or scr. Transduced cells were sorted 4 days post transduction and incubated in complete medium for 48 h followed by Annexin V-FITC/PI staining. 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 = 2). The data were analyzed by two-sided T-test. *, p < 0.05, ***, p < 0.001. (F,G) BL cell lines were transduced with F1sh, with a vector expressing F1wob and F1sh (F1sh-F1wob), or with scr control. (F) Cells were sorted on day 4 and the expression of FOXO1 was analyzed by immunoblot. TUBB served as loading control. A representative of 2 independent experiments is shown. (G) The percentage of RFP+ cells was measured every 3 days using flow cytometry. First measurement was performed 4–5 days post transduction and the percentage of RFP+ cells was set as 100. Data are shown as mean ± SD (n = 3).