Baicalin increases the intracellular Ca²⁺ levels in HepG2 cells.

<p>Traces show the increases in intracellular Ca²⁺ levels in response to application of ionomycin (A) and baicalin (B), as well as application of HBSS solution (C) in HepG2 cells loaded with Ca²⁺ indicator fura 2-AM. Intracellular Ca²⁺ levels were estimated as the ratio of the signals (F₃₄₀/F₃₈₀). (D) Histogram summarizes the changes of intracellular Ca²⁺ levels (ΔF₃₄₀/F₃₈₀) measured after application of HBSS (open bars, n = 3), ionomycin (peak values, hatched bars, n = 3), and baicalin (solid bars, n = 3). * <i>p</i><0.05 and ** <i>p</i><0.01 compared to HBSS control.</p

Effects of baicalin on ROS and ATP production.

<p>HeLa (A) and HepG2 (B) cells were exposed to various concentrations of baicalin for indicated times. Intracellular ROS production quantified using the fluorescent probe DCFDA (top), and the cell viability was determined by MTT test (bottom). Data were expressed as mean ± SE relative to vehicle control from at least four independent experiments. * <i>p</i><0.05 compared to vehicle control.</p

Inhibition of CaMKKβ blocks baicalin-induced AMPKα and ACC phosphorylation in HeLa cells.

<p>(A) Representative Western blots showing the effect of the CaMKKβ inhibitor STO-609 on baicalin-induced phosphorylation of AMPKα at Thr-172 (pAMPKα) and phosphorylation of ACC at Ser-79 (pACC) as well as total AMPKα, ACC and CaMKKβ protein expression. Cells were treated with 5 µM baicalin for 1 hr or 1 µM ionomycin for 5 min in the absence or presence of 10 µg/mL STO-609. (B) Histogram represents the fold change in the pAMPKα/AMPKα or pACC/ACC ratio from at least three independent experiments. * <i>p</i><0.05 compared to respective untreated control; ^#<i>p</i><0.05 and ^##<i>p</i><0.01 compared to respective treatment group in the absence of STO-609.</p

Inhibition of CaMKKβ prevents baicalin-induced reduction in intracellular lipid accumulation in HeLa cells caused by oleic acid.

<p>Cells were treated with 1 and 5 µM baicalin in the presence of 0.4 mM oleic acid for 24 hr without (open bars) or with pre-incubated with 10 µg/mL STO-609 (solid bars). The control cells were incubated with 1.76% bovine serum albumin (BSA) for 24 h. Intracellular triglyceride (TG, A) and intracellular cholesterol (TC, B) were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047900#s4" target="_blank">Material and Methods</a>. The histogram represents the mean of the percentage of the vehicle control ± SE from at least three independent experiments. * <i>p</i><0.05 compared to BSA control; ^#<i>p</i><0.05 compared to untreated oleic acid control.</p

Effects of inhibition of CaMKKβ and intracellular Ca²⁺ on baicalin-induced phosphorylation of CaMKI in HeLa cells.

<p>HeLa cells were treated with 5 µM baicalin for 1 hr or 1 µM ionomycin for 5 min in control medium (open bars), or in the presence of 10 µg/mL of the CaMKKβ inhibitor STO-609 (solid bars, A), Ca²⁺-free medium containing Ca²⁺ chelators EDTA and EGTA (solid bars, B), or medium containing 2 µM thapsigarging to deplete intracellular Ca²⁺ stores (solid bars, C). Phosphorylation of CaMKI at Thr-177 (pCaMKI) as well as total CaMKI and β-actin protein expression were measured by Western blotting. Histograms represent the fold change in the pCaMKI/CaMKI ratio from at least three independent experiments. * <i>p</i><0.05 and ** <i>p</i><0.01 compared to control; ^#<i>p</i><0.05 and ^##<i>p</i><0.01 compared to respective treatment group in control medium. Representative Western blots are shown.</p

Baicalin increases the phosphorylation of AMPKα and ACC without effect on ATP in HeLa cells.

<p>Levels of AMPKα phosphorylated at Thr-172 (pAMPKα) and of AMPK substrate ACC phosphorylated at Ser-79 (pACC) as well as total AMPKα and ACC were determined by Western blotting. Cells were treated with baicalin or AICAR (500 µM) for the indicated times without (A) or with (B) pre-treatment of the AMPK inhibitor compound C (40 µM). Histograms represent the fold change in the pAMPKα/AMPKα or pACC/ACC ratio from at least three independent experiments. (C) Cells were incubated with baicalin for indicated times and ATP levels were measured. All values are the mean ± SE for three independent experiments. * <i>p</i><0.05 compared to respective control; # <i>p</i><0.05 compared to respective baicalin group in the absence of compound C. Representative Western blots are shown.</p

Baicalin increases the phosphorylation of AMPKα and ACC without effect on ATP in HepG2 cells.

<p>(A) Levels of AMPKα phosphorylated at Thr-172 (pAMPKα) and of AMPK substrate ACC phosphorylated at Ser-79 (pACC) as well as total AMPKα and ACC were determined by Western blotting. Cells were treated with various concentrations of baicalin or AICAR (500 µM, 2 hr) for the indicated times. Histograms represent the fold change in the pAMPKα/AMPKα or pACC/ACC ratio from at least three independent experiments. (B) Cells were incubated with baicalin for indicated times and ATP levels were measured. All values are the mean ± SE for two-three independent experiments. * <i>p</i><0.05 compared to respective control. Representative Western blots are shown.</p

Effects of inhibition of CaMKKβ on baicalin-induced phosphorylation of AMPKα and phosphorylation of CaMKI in HepG2 cells.

<p>Cells were treated with 5 µM baicalin for 1 hr or 1 µM ionomycin for 5 min in control medium (open bars) or in the presence of 10 µg/mL of the CaMKKβ inhibitor STO-609 (solid bars). Representative Western blots for phosphorylation of AMPKα at Thr-172 (pAMPKα) and phosphorylation of CaMKI at Thr-177 (pCaMKI), and expression of total AMPKα, CaMKI, CaMKKβ and LKB1 (A). Histograms represent the fold change in the pAMPKα/AMPKα (B) or pCaMKI/CaMKI (C) ratio from at least three independent experiments. * <i>p</i><0.05 compared to control; ^#<i>p</i><0.05 compared to respective treatment group in control medium. Representative Western blots are shown.</p

Baicalin increases the intracellular Ca²⁺ levels in HeLa cells.

<p>Traces show the increases in intracellular Ca²⁺ levels in response to application of ionomycin (1 µM) (A), baicalin (5 µM) in HBSS solution (B) and after depletion of intracellular Ca²⁺ stores by thapsigargin (Tha, D), as well as application of HBSS solution (C) in HeLa cells loaded with Ca²⁺ indicator fura 2-AM. Intracellular Ca²⁺ levels were estimated as the ratio of the signals (F₃₄₀/F₃₈₀). (E) Histogram summarizes the changes of intracellular Ca²⁺ levels (ΔF₃₄₀/F₃₈₀) measured after application of HBSS (open bars, n = 3), ionomycin (peak values, hatched bars, n = 3), baicalin in HBSS (solid bars, n = 7) or in the presence of Tha (solid bars, n = 3). * <i>p</i><0.05 and ** <i>p</i><0.01 compared to HBSS control; <i>p</i><0.001 compared between two groups as indicated.</p

Role of intracellular Ca²⁺ in baicalin-induced AMPKα phosphorylation.

<p>HeLa cells were treated with 5 µM baicalin for 1 hr or 1 µM ionomycin for 5 min in control medium (open bars), Ca²⁺-free medium containing Ca²⁺ chelators EDTA and EGTA (solid bars, A), or medium containing 2 µM thapsigarging to deplete intracellular Ca²⁺ stores (solid bars, B). Phosphorylation of AMPKα at Thr-172 (pAMPKα), total AMPKα and β-actin expression were measured by Western blotting. Histograms represent the fold change in the pAMPKα/AMPKα ratio at least three independent experiments. * <i>p</i><0.05 compared to control; ^#<i>p</i><0.05 compared to respective treatment group in control medium. Representative Western blots are shown.</p