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. 2019 Sep 18;11(9):1394. doi: 10.3390/cancers11091394

Figure 4.

Figure 4

The nuclear localization of FOXO3 by inhibition of JAK2. (A) Cells were treated with DMSO vehicle control or the indicated concentration of AZD1480 (0, 2.5, 5, and 10 μM) for 48 h, washed with PBS, trypsinized, and lysed in the cytoplasmic fractional buffer. After separating the cytoplasmic fraction, the remaining pellet was washed with washing buffer and lysed with nuclear fractional buffer, and the supernatant was collected for the nuclear fraction. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Lamin B1 were used for a gel loading control for the cytoplasmic and nuclear protein fractions, respectively. (B) Pharmacological inhibition of JAK2 induces the nuclear localization of FOXO3 in A498 and ACHN. Cells were treated with DMSO vehicle control or AZD1480 (5 μM) for 2 h and then incubated with a primary antibody against FOXO3 followed by Alexa 594-conjugated anti-mouse secondary antibody. The nuclei were counterstained by 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images were captured with a confocal microscope. Scale bar: 5 μm (C) Pharmacological inhibition of JAK2 increases the protein stability of FOXO3 in 293T cells transfected with pECE Flag-FOXO3 and pCMV3-C-HA- JAK2 plasmid DNA via a proteasome-mediated pathway. Next, 293T cells were treated with DMSO (control vehicle), cycloheximide (CHX, 20 μg/mL), or MG-132 (20 μM) with/without AZD1480 (5 μM) for the indicated time and then cell lysates were resolved on the SDS-PAGE and immunoblotted by the anti-FOXO3 antibody. β-actin was used for the loading control. (D) Pharmacological inhibition of JAK2 decreases the ubiquitination of FOXO3 in 293T cells transfected with pECE Flag-FOXO3 and pCMV3-C-HA-JAK2 plasmid DNA. 293T cells were treated with DMSO (control vehicle) or MG132 (20 μM) with/without AZD1480 (5 μM) for the indicated time and then cell lysates were immunoprecipitated with anti-FOXO antibody and blotted with anti-ubiquitin, Flag, and FOXO3 antibody. The heavy chain of IgG was used for the loading control. For input analysis, cell lysates were resolved on the SDS-PAGE and immunoblotted by anti- JAK2, pJAK2, and FOXO3 antibody. β-actin was used for the loading control.