AMPK activation was decreased in LCC2 and LCC9 cells compared to MCF7S cells.

<p>(A) Cells were treated with 1 mM AICAR for 60 min. p-AMPK (Thr172), p-TSC2 (Ser1387), and HIF-1α was detected by western blotting. β-Actin was served as a loading control. The data shown are representative results for at least 3 independent western blot analyses. Band density was quantified using MultiGauge. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001 vs. no treatment of AICAR and ^#, <i>P</i> < 0.05; ^##, <i>P</i> < 0.01; ^###, <i>P</i> < 0.001 vs. MCF7S control cells. (B) AICAR-treated cells were assayed for lactate production. (C) Cells treated with AICAR were treated with 4-OHT (5 μM) for 3 days and cell survival of MCF7S, LCC2, and LCC9 cells was measured by XTT assay. The data represent the mean ± SD of representative results for 3 independent lactate assays. *, <i>P</i> < 0.05; ***, <i>P</i> < 0.001.</p

HIF-1α is upregulated in LCC2 and LCC9 cells.

<p>(A) HIF-1α activity in MCF7S, LCC2 and LCC9 cells was measured by EMSA. (B) Endogenous HIF-1α and LDHA protein levels were measured by western blotting. β-Actin was used as a loading control. (C) The mRNA level of HIF-1α was measured by quantitative RT-PCR using the β-actin gene as an internal control. (D) LDHA was downregulated in MCF7S, LCC2, and LCC9 cells transfected with HIF-1α siRNA. siRNA (20nM) was treated in three cell lines for 48 h. C, control siRNA; H, HIF-1α siRNA (E) HIF-1α was overexpressed by treatment of CoCl₂ (50 μM, 24 h) in each cell line. Elevation of HIF-1α induced increase in LDHA expression levels. The data shown are representative results of 3 independent experiments. Band density of each immunoblot was quantified using MultiGauge. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p

Increased Akt/mTOR signaling induces HIF-1α activity and aerobic glycolysis.

<p>(A) VHL protein levels, as well as the activity of Akt/mTOR and its downstream targets measured by western blotting using β-actin as a loading control. (B) After treatment with specific inhibitors of Akt (LY294,002, 40 μM) and mTOR (rapamycin, 30 nM) for 4 and 16 h, respectively, HIF-1α and Akt/mTOR protein expressions were measured by western blotting in LCC2 cells. β-Actin was used as a loading control. Band density of each immunoblot with triplicate was quantified using MultiGauge. (C) Rapamycin at 30 nM was used to treat LCC2 cells for 16 h. HIF-1α activity was measured using EMSA in LCC2 cells. The data represent results of 3 independent experiments. (D) Lactate accumulation was measured by ¹H NMR quantification after treatment of rapamycin (30 nM, 16 h). *, <i>P</i> < 0.05.</p

Somatic mtDNA mutations in MCF7S, LCC 2, and LCC9 cells.

<p>*D-L, D-loop; 12S, 12S ribosomal RNA; ND2, 5 NADH dehydrogenase subunit 2, 5; ATPase, ATP synthase F0 subunit 8; COX3, cytochrome C oxidase subunit III; CYTB, cytochrome B; and NPCL, non-protein coding locus</p><p>Somatic mtDNA mutations in MCF7S, LCC 2, and LCC9 cells.</p

Lactate production and glucose consumption were elevated in LCC2 and LCC9 cells.

<p>(A) Cell proliferation rate of MCF7S, LCC2 and LCC9 cells was measured by XTT assay in a time-dependent manner. The data represent the mean ± SD of representative results for 3 independent assays. (B) Viability of MCF7s, LCC2 and LCC9 cells was measured by XTT assay 24 h after the treatment various concentration of 3-BrPA. The data represent the mean ± SD of representative results for 3 independent assays. (C) The amount of ¹³C-labeled lactate was determined in each cell line by ¹H NMR quantification. ¹³C-labeled lactate derived from glycolysis was traced and quantified for each cell line. Spectra were normalized to the cell dry weight and an NMR scaling factor. (D) The amount of total lactate in the cell extracts was quantified through GC-MS analysis. The vertical axis represents the amount of total lactate (μM) per dry weight of protein (g). (E) ³H-glucose was used to treat each cell line for 2 h. Glucose uptake was expressed as counts per minute per milligram of protein (cpm/min/mg protein). (F) Inhibition of hexokinase-2 (HK-2) repressed cell proliferation, especially in LCC2 and LCC9, compared with MCF7S. Control siRNA (Consi) and HK-2 siRNA (HK2si) were transfected into each cell line with 20 nM for 24 h and cell proliferation was measured after 48 h transfection. The data represent the mean ± SD of 3 independent experiments that were performed in triplicate. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p

Akt/mTOR/HIF-1α axis was specifically suppressed by knockdown of hexokinase-2 (HK-2) glycolytic enzyme in LCC2 and LCC9 cells.

<p>(A) Cells were transfected with control siRNA (Consi) or HK-2 siRNA (HK2si) at 20 nM for 48 h followed by treatment of 4-OHT (10 μM, 48 h). (B) Cells were treated with 3-BrPA (50 μM) for 4 h and followed by treatment of 4-OHT (10 μM, 48 h). Western blotting was performed for the lysates prepared from treated cells. β-Actin was served as a loading control. The data shown are representative results for at least 3 independent experiments. Band density was quantified using MultiGauge. *, <i>P</i> < 0.01; **, <i>P</i> < 0.001 vs. Consi transfected cells.</p