Figure 3.
Figure 3. Nuclear FOXO1 promotes lymphoma growth. (A) Experimental scheme of CRISPR/Cas9-mediated FOXO1 ablation in murine BL cells. After transient expression of Cas9 and the Foxo1 gRNA_d, which highly efficiently targets wt and mt Foxo1, reporter-positive cells were FACS-based sorted and the bulk of tumor cells analyzed in panels B-C. CRISPR/Cas9 target sequences are given in blue (protospacer) and red (PAM). (B) Foxo1 T7EI assay in CRISPR/Cas9-modified mouse BL cells at indicated time points after FACS-based sorting of mCherry-positive cells. Two cell lines characterized by a heterozygous T24 mutation (mouse BL#19 and #82) were analyzed as well as 2 cell lines exclusively expressing wt FOXO1 (mouse BL#81 and #88). Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7EI. The calculated rate of indel mutagenesis after quantification of band intensities is shown in the graphs. (C) Foxo1 sequence analysis after CRISPR/Cas9 editing in mouse BL cell lines (mouse BL#19 and #82) at indicated time points after FACS-based sorting. Pie charts depict the distribution of CRISPR edited (targeted) vs untargeted sequences. The total number of sequences analyzed is given in the center of the chart. (D) Experimental setup for stable FOXO1 ablation in human BL cells. Cas9 coupled with an mCherry fluorescent reporter was introduced to the cells by lentiviral infection. Single mCherry-positive cells were FACS-based sorted, and stable expressing Cas9 clones were further expanded. In a second step, Cas9-positive clones were infected with a lentiviral construct encoding FOXO1 gRNA coupled to a BFP fluorescent reporter. mCherry- and BFP-coexpressing cells were sorted and Sanger sequencing was performed in genomic DNA extracted from the bulk of tumor cells at indicated time points. The protospacer (PAM) sequence is given in blue (red) as in panel A. (E) FOXO1 sequence analysis after CRISPR/Cas9 editing in human BL cell lines (Namalwa, BL2, and CA46) at indicated time points after FACS-based sorting. CRISPR-edited sequences were analyzed and the distribution of “out-of-frame” vs “in-frame“ sequences is shown in the pie charts. The total number of sequences analyzed is given in the center of the chart.

Nuclear FOXO1 promotes lymphoma growth. (A) Experimental scheme of CRISPR/Cas9-mediated FOXO1 ablation in murine BL cells. After transient expression of Cas9 and the Foxo1 gRNA_d, which highly efficiently targets wt and mt Foxo1, reporter-positive cells were FACS-based sorted and the bulk of tumor cells analyzed in panels B-C. CRISPR/Cas9 target sequences are given in blue (protospacer) and red (PAM). (B) Foxo1 T7EI assay in CRISPR/Cas9-modified mouse BL cells at indicated time points after FACS-based sorting of mCherry-positive cells. Two cell lines characterized by a heterozygous T24 mutation (mouse BL#19 and #82) were analyzed as well as 2 cell lines exclusively expressing wt FOXO1 (mouse BL#81 and #88). Arrows indicate the expected positions of DNA bands cleaved by mismatch-sensitive T7EI. The calculated rate of indel mutagenesis after quantification of band intensities is shown in the graphs. (C) Foxo1 sequence analysis after CRISPR/Cas9 editing in mouse BL cell lines (mouse BL#19 and #82) at indicated time points after FACS-based sorting. Pie charts depict the distribution of CRISPR edited (targeted) vs untargeted sequences. The total number of sequences analyzed is given in the center of the chart. (D) Experimental setup for stable FOXO1 ablation in human BL cells. Cas9 coupled with an mCherry fluorescent reporter was introduced to the cells by lentiviral infection. Single mCherry-positive cells were FACS-based sorted, and stable expressing Cas9 clones were further expanded. In a second step, Cas9-positive clones were infected with a lentiviral construct encoding FOXO1 gRNA coupled to a BFP fluorescent reporter. mCherry- and BFP-coexpressing cells were sorted and Sanger sequencing was performed in genomic DNA extracted from the bulk of tumor cells at indicated time points. The protospacer (PAM) sequence is given in blue (red) as in panel A. (E) FOXO1 sequence analysis after CRISPR/Cas9 editing in human BL cell lines (Namalwa, BL2, and CA46) at indicated time points after FACS-based sorting. CRISPR-edited sequences were analyzed and the distribution of “out-of-frame” vs “in-frame“ sequences is shown in the pie charts. The total number of sequences analyzed is given in the center of the chart.

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