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. 2022 Dec 15;132(24):e160767. doi: 10.1172/JCI160767

Figure 3. FOXO1 acts upstream of the MCL lineage regulatory TF hierarchy.

Figure 3

(A) Immunoblot analysis of CCMCL1 cells transduced with EBF1-V5 (left), 3× FLAG–tagged IRF4 (middle), or 3× FLAG–tagged PAX5 (right). (B) Competition-based proliferation assays of sgRNAs against FOXO1 in Cas9-transduced CCMCL1 cells expressing mock control, EBF1-V5, or 3× FLAG–tagged IRF4 or PAX5. (C) Competition-based proliferation assays of sgRNAs against EBF1, IRF4, or PAX5 in control and FOXO1r#1-transduced (resistant for sgRNA#1 of FOXO1) CCMCL1 cells. In B and C, data represent mean ± SEM (n = 3). Results are representative of 3 independent experiments. Statistical analysis was performed using 2-tailed unpaired Student’s t test. The day 13 values of each cell line responding to the same sgRNA were respectively compared. **P < 0.001 ***P < 0.0005, ****P < 0.0001. (D) Immunoblot analysis of CCMCL1 (left) or JEKO1 (right) cells depleted of FOXO1. Lysates were prepared at day 3 after infection of indicated sgRNAs targeting FOXO1. (E–H) Immunoblot and RT-qPCR analysis of EBF1, IRF4, or PAX5 induction in FOXO1-transduced HEL (E and F) or THP1 cells (G and H). Cell lysates and total RNA were prepared at indicated time points after infection of FOXO1-encoding lentivirus. In F and H, data represent mean ± SEM (n = 3). Results are representative of 3 independent experiments. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.