Fig 2: A2BR promotes FOXO3 binding and expression of pluripotency factor genes through activation of p38 MAPK. A, MDA-MB-231 cells were treated with 10 nM paclitaxel (P), alone or in combination with 10 μM alloxazine (A2BRi), 2.5 μM H-89 (PKAi), 1 μM Gö6983 (PKCαi), 1 μM rottlerin (PKCδi), 1 μM MK-2206 (AKTi), or 5 μM SB203580 (p38i), for 72 h. RT-qPCR assays were performed and log2 fold change of mRNA expression of pluripotency factors (vs. P) is presented as a heat map. B, MDA-MB-231 NTC or A2BR knockdown subclones were treated with vehicle (V) or 10 nM paclitaxel (P) for 72 h and immunoblot assays were performed. C, MDA-MB-231 NTC or A2BR knockdown subclones were treated without (-) or with (+) 5 μM adenosine for 72 h and immunoblot assays were performed. D, MDA-MB-231 cells were treated with 10 nM paclitaxel, 5 μM SB203580, or both for 72 h. ChIP was performed using FOXO3 antibody followed by qPCR with primers flanking FOXO3 binding sites in the NANOG, SOX2 and KLF4 gene (mean ± SEM; n = 4); *p < 0.05, **p < 0.01, ***p < 0.001 vs. V; ##p < 0.01, ###p < 0.001 vs. P. E-H, MMTV-PyMT transgenic mice were treated with V, P (5 mg/kg, days 0, 5, and 10), LY2228820 (LY; 10 mg/kg, days 0-13), or P + LY, when tumors reached a cumulative volume of 150 mm3. Tumor volumes were measured every 2-3 days (E). Tumors were harvested on day 13 for ALDH (F), mammosphere (G), and RT-qPCR (H) assays. Data are shown as mean ± SEM (n = 5); *p < 0.05, **p < 0.01, ***p < 0.001 vs. V; #p < 0.05, ##p < 0.01 vs. P; ns, not significant.

Fig 3: Effects of FOXO3 on DLBCL cells were abolished by CNN3. (A) The mRNA level of CNN3 in DB cells. (B) Cell viability was detected using CCK-8 assay. (C and D) Cell cycle distribution was analyzed using flow cytometry. (E and F) Apoptotic levels were measured using flow cytometry with Annexin V and PI staining. (G) FOXO3-regulated CNN3 promoted the tumorigenesis of DLBCL cells by enhancing proliferation and cell cycle transition, and suppressing apoptosis. *P<0.05, **P<0.01 and ***P<0.001 vs. the pcDNA3.1 group; ^P<0.05, ^^P<0.01 and ^^^P<0.001 vs. the FOXO3+pcDNA3.1 group. FOXO3, forkhead box O3; DLBCL, diffuse large B-cell lymphoma; CNN3, calponin 3; CCK-8, Cell Counting Kit-8; PI, propidium iodide.

Fig 4: Inhibition of Akt activation reduces FOXO3 sequestration in the cytoplasm and proliferation of SVZ-derived NPCs. a Cytoplasmic fractions of SVZ-derived NPCs from the ConV- or EcoHIV-infected mice were separated on SDS-PAGE to evaluate the expression of phosphorylated Akt. GAPDH was used for data normalization. Unpaired t test, N=3~4 per group. *, p<0.05. b SVZ-derived NPCs from METH- and EcoHIV-exposed mouse brain were treated with 10 μM LY294002 for 24 h to evaluate the level of pAkt, pErk, and FOXO3 separately into the cytoplasmic (Cyto F) and nuclear (Nuc F) fractions. Lamin A/C was used as a marker for Nuc F and GAPDH was for Cyto F. c Quantitative results from (b). Unpaired t test, N=4 per group. **, p<0.01. d SVZ-derived NPCs were treated with LY294002 (10 μM) for 24h and in the presence of EdU (10 μM) during the last 30 min. After PBS wash, cells were fixed, permeabilized, and stained with TAMRA azide (red) for 30 min. Nuclei were stained with DAPI and images were taken by a confocal microscope. Note the apparent decrease in proliferating cells as the result of LY294002 treatment. Scale bar, 100 μm. e The percentage of EdU-positive cells from (d) were calculated and compared to the total number of nuclei (mean ± SEM). N=4 individual slides per group and 5–8 visual fields per slide. *, p<0.05

Fig 5: METH exposure and EcoHIV infection affect the expression of the CXCL12-CXCR4-FOXO3 axis. a Differentially expressed mRNAs of representative FOXO-targeted genes by METH exposure and/or EcoHIV infection in NPCs from Fig. 4. The upper plots are representative genes regulated by the FOXO family of transcription factors and the lower plots are representative FOXO3-regulated genes. b Representative immunoblots of CXCL12, phosphorylated CXCR4 (pCXCR4), and FOXO3. GAPDH was used as a loading control. c Quantitative results from (b). The intensity of the bands corresponding to CXCL12, pCXCR4, and FOXO was normalized to the corresponding GAPDH intensity, and the mean and SEM were calculated. Two-way ANOVA was used for statistical analysis. N=4–6 per group. *, p<0.05 and **, p<0.01. d Representative images of FOXO3- and Sox2-positive cells in the SVZ. Mice were exposed to METH and/or infected with EcoHIV as in Fig. 2b. Frozen brain sections were immunostained for FOXO3 (green foci) and Sox2 (red) and examined under the confocal microscope. Scale bar = 20 μm; z-stacked images