Fig 2: Genetic and pharmacological blockage of PFKFB3 inhibits the bone resorption activity of osteoclasts. A, B, Mature osteoclasts from BMMs were seeded in Corning osteo assay strip wells and infected with the adenovirus carrying PFKFB3 shRNA or the control adenovirus, then cultured for 2 d with M-CSF and RANKL supplementation. F-actin staining (A) or pit formation (B) assays were performed. C, D, Mature osteoclasts from BMMs were seeded in Corning osteo assay strip wells and treated with different concentrations of PFK15 for 2 d in the presence of M-CSF and RANKL. F-actin staining (C) or pit formation (D) assays were conducted. *P < .05, **P < .01 vs control. Data are presented as means ± SD of three independent experiments. Original scale bars, 250 µm

Fig 3: ATM signaling stimulates PFKFB3 recruitment upon DNA damage induction. a Confocal analysis of DNA damage, ?H2AX, and nuclear localization of PFKFB3 in U2OS cells following treatment with inhibitors (6 h) as indicated, subjected to IR (6 Gy), or left untreated, n = 3 independent experiments. Scale bar, 10 µm. b Scatter dot plot representing the PFKFB3 nuclear intensity in (a) as quantified using CellProfiler, n > 100 cells/treatment. ***P < 0.001; one-way ANOVA analysis. c Scatter dot plot representing the ?H2AX nuclear intensity in (a) as quantified using CellProfiler, n > 100 cells/treatment. **P < 0.01, ***P < 0.001; one-way ANOVA analysis. d Bars representing the percentage of PFKFB3 and ?H2AX foci that co-localize in (a) as quantified using CellProfiler, n > 100 cells/treatment. Data are displayed as means ± SEM, ***P < 0.001, one-way ANOVA was used to calculate statistical significance

Fig 4: PFKFB3 synthesizes F2,6BP, which in turn activates PFK-1 and glycolysis in the cytosol and Cdk1 in the nucleus. Taken together, the data presented in the initial report of the functions of nuclear PFKFB3 and this article support a model whereby PFKFB3 in the cytosol regulates glucose metabolism, whereas PFKFB3 in the nucleus regulates Cdk1 activity and p27 protein levels. Both glucose metabolism and Cdk1 activity are required for growth and resistance to apoptosis

Fig 5: Dendropanax trifidus (DT) sap-induced signalings in C2C12 muscle cells. (A,B) Western blot images of C2C12 cells treated with vehicle control or 25 µg/mL DT for 1 h or 24 h and probed with pAMPK/AMPK antibodies (A) and AMPK-downstream signaling antibodies related with lipid metabolism (pACC, ACC, pHSL, HSL, ATGL); glucose metabolism (PFKFB3); autophagy and mitochondrial homeostasis (pULK1 and ULK1), and b-Actin (B). Three independent biological replicates are provided here. (C–J) Image analysis of western blot membranes of (B). Relative ratios for the indicated signals were calculated as follows: pACC/ACC (C); ACC/b-Actin (D); pHSL/HSL (E); HSL/b-Actin (F); ATGL/b-Actin (G); PFKFB3/b-Actin (H); pULK1/ULK1 (I); ULK1/b-Actin (J). 30 µg protein was loaded per lane. N = 3. Student’s t-test: * p < 0.05, comparison with vehicle control. Bars indicate mean ± S.E.M.