Fig. 6: Expression of FOXO4 is regulated by hTERT phosphorylated on T249.

a MA plot of CAGE peaks differentially expressed between the T249A-CRISPR and Control-CRISPR (left panel). x- and y-axes indicate average expression levels and fold changes with triplicates, respectively. Individual dots represent CAGE peaks corresponding to promoters defined by the FANTOM5 project⁷⁷. CAGE peaks with statistical significance are indicated by pink and light blue for higher and lower expression in the T249A-CRISPR, respectively. FOXO4 are indicated with red on MA plot and the positions of each FOXO4 CAGE peak are visualized by IGV (right panel). b The endogenous proteins were detected by anti-FOXO4 mAb in T249A-CRISPR while not in Control-CRISPR. β-actin: an internal control. c Immune complexes were isolated with 10E9-2 and associated RNAs were subjected to RT-PCR. d Transfection of asRNA complementary to the first exon of FOXO4 or firefly luciferase mRNA. Cellular RNAs were subjected to RT-PCR. “RT” indicates reverse transcriptase reaction. The relative expressions are noted below the panel (for c, d). e Immunoblotting of ectopically overexpressed FOXO4 (left panel) and cell proliferation (right panel); infected with pBABE-hygro (pBh) (black) and pBh-FOXO4 (red) retroviruses. The cell proliferation assay was done in triplicate and data are shown as the mean ± SD. Asterisk: statistically significant values (p < 0.05 by Student’s t-test, two-sided). f Tumor appearances generated from subcutaneous injection in eight mice (upper panel). The volume curves of tumors are demonstrated as the mean ± SEM (n = 8) (lower panel). Asterisk: statistically significant values (p = 0.0092 by unpaired t-test, two-sided). g Immunoblotting of FOXO4 (upper panel). Cell proliferation assay (lower panel) was done in triplicate and data are shown as the mean ± SD. Asterisk: statistically significant values (p < 0.05 by Student’s t-test, two-sided). h Tumors were established from subcutaneous injection in seven mice (upper panel). Tumor-volume curves of xenografts are shown as the mean ± SEM (n = 7) (lower panel). Asterisk: statistically significant values (p < 0.05 by unpaired t-test). Control-CRISPR (black), T249A-CRISPR+shGFP (red), T249A-CRISPR+shFOXO4-1 (blue), and T249A-CRISPR + shFOXO4-2 (green) (for g, h). Experiments were repeated three times (for b) and twice (for c–e (left panel), g (upper panel)) with similar results. Source data are provided in the Source Data file.