FOXO1 knockdown downregulates CCND3 transcription. (A-B) RS4;11 and 018Z BCP-ALL cell lines were transduced with lentiviral vectors expressing scrambled (Scr) or FOXO1-specific shRNA-81. Cells were sorted by FACS on day 5 after transduction and used for isolation of total RNA and protein. Downregulation of CCND3 mRNA (A) and protein (B) by shFOXO1-81 were measured by qRT-PCR (N = 2) and immunoblot, respectively. A representative of 2 independent protein isolations is shown. (C) Efficient knockdown of CCND3. BCP-ALL cell lines were transduced with a lentiviral vector expressing scrambled (Scr) or targeting CCND3 (shC3) shRNA. The RFP⁺ cells were sorted by FACS on day 3 after transduction and CCND3 expression was assessed by immunoblot. A representative of 2 independent experiments is shown. (D) Antileukemia effect of CCND3 knockdown. The dynamic of the RFP⁺ population was measured every 3 days starting from the day 3 after infection. The proportion of RFP⁺ cells on the first day of measurement (day 3) was taken as 100%. Data represent mean ± SD of 3 independent transductions. (E) CCND3 knockdown decreases expression of the proliferation marker MKI67. Cells were sorted on day 3 after transduction and used for protein isolation. Repression of MKI67 was demonstrated by immunoblot. (F) CCND3 knockdown induces G₁ cell cycle arrest. Transduced cells were sorted on day 3 after transduction, and cell cycle was analyzed by PI staining on the next day after sorting (N = 2). (G) CCND3 knockdown downregulates RB1 phosphorylation and MYC expression, induces CASP3 cleavage, and increases CDKN1B expression. Cells were sorted on day 3 after transduction and the protein expression and phosphorylation was measured by immunoblot (N = 2). (H-I) CCND3 overexpression protects BCP-ALL cells from the cytotoxic effect of FOXO1 knockdown. 018Z and NALM-6 cells were transduced with SF-LV-cDNA-eGFP empty vector (EV) or with SF-LV-CCND3-expressing vector. In 4 to 6 days, the cells were transduced with shF1-81 or scr vectors expressing the fluorescent marker RFP. (H) For analysis of CCND3 protein expression, the cells were sorted by FACS on day 3 after the second transduction. (I) The percentage of the GFP⁺/RFP⁺ population was measured at indicated time points after the second transduction. Data are shown as mean ± SD (N = 2). (J) CCND3 overexpression restores RB1 phosphorylation and MYC expression, but not the decrease of S6 phosphorylation induced by FOXO1 knockdown (N = 2). The 018Z cells overexpressing CCND3 of control vector were transduced with vectors expressing shF1-81 or scr and sorted on day 4 after transduction. The protein expression and phosphorylation status was measured by immunoblot (N = 2). *P < .05; **P < .01; ***P < .001; ****P < .0001.