Effect of YLT322 on the expression levels of apoptosis-related proteins and the mitochondrial membrane potential (ΔΨ) of HepG2 cells.

<p><b>A.</b> The expression levels of pro-caspase-8, -3, -9, and their activated forms in HepG2 and Bel-7402 cells treated with YLT322 for 48 hours as assayed by western blot. <b>B.</b> HepG2 cells were treated with 2 µM YLT322 alone or in combination with Z-VAD-FMK (a general caspase inhibitor), Ac-LEHD-FMK (caspase-9 inhibitor) or Ac-IETD-FMK (caspase-8 inhibitor). Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001). <b>C.</b> The mitochondrial membrane potential (ΔΨ) was detected by RH123 staining after treatment with YLT322 at varying concentrations of 0 µM (Dark blue), 0.5 µM (blue), 1 µM (orange), 2 µM (green) for 12 to 48 hours (Bars, SD; Column, mean; n, 3;*p<0.05; **p<0.01; ***p<0.001). <b>D.</b> The level of cytochrome <i>c</i> in the cytosol and mitochondrial fraction was analyzed by western blot analysis after incubation of cells with YLT322 for 48 hours. Expression of COX-4 served as the loading control for the mitochondria fraction. <b>E.</b> The protein levels of Bcl-2 and Bax were detected by western blot in HepG2 cells treated with YLT322 for 48 h. <b>F.</b> The mRNA level of Bax was examined by real-time RT-PCR in HepG2 cells treated with YLT322 for 48 h. (Bars, SD; Column, mean; n, 3;*p<0.05; **p<0.01; ***p<0.001). <b>G.</b> The expression levels of Akt, p44/42 MAPK and their phosphorylated forms were assayed by western blot in HepG2 cells treated with YLT322. <b>H.</b> HepG2 cells were treated with 2 µM YLT322 alone or in combination with LY294002 (PI3K/AKT inhibitor) or PD98059 (MEK/ERK inhibitor). Data shown are representative of three independent experiments.</p

The effect of YLT322 on cell morphology and viability of cancer cells.

<p><b>A.</b> Flow cytometric analysis of PI-stained HCC cell lines, including HepG2, SMMC-7721, Bel-7402 and Bel-7404, after treatment with 2 µM YLT322 for 48 hours. <b>B.</b> Statistical results of apoptosis assays presented as surviving cells (percentage of untreated control). Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001). <b>C.</b> Fluorescence microscopy analysis of Hoechst 33342-stained HepG2 cells after incubation with varying concentrations of YLT322 (0 µM (a), 0.5 µM (b), 1 µM (c), 2 µM (d)) for 24 hours (×40). <b>D.</b> Flow cytometric analysis of cells stained with Annexin V-FITC/PI after treatment with various concentrations (0 µM, 0.5 µM, 1 µM, 2 µM) of YLT322 for 12 to 48 hours.</p

Inhibition of cell growth and colony formation in human cancer cell lines by YLT322 <i>in vitro</i>.

<p><b>A.</b> Cells (a:HepG2; b:Bel-7402; c: Bel-7404;d:SMMC-7721) were treated with concentrations of YLT322 ranging from 0.15 to 5.0 µM, for 12 to 48 hours and cell viability was determined by MTT assay . Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001) <b>B.</b> Effects of varying concentrations of YLT322 on colony formation of HepG2 and Bel-7402 cells after two weeks treatment. <b>C.</b> Statistical results of colony-forming assays presented as surviving colonies (percentage of untreated control). Data are expressed as mean ± SD. for at least 3 independent experiments. (*p<0.05; **p<0.01; ***p<0.001).</p

The chemical structure of YLT322.

<p>The chemical structure of YLT322.</p

The proliferation inhibition of YLT322 in human tumor cell lines.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063900#pone-0063900-t001" target="_blank">Table 1</a>. IC₅₀ values (µM) for inhibition of cell proliferation by 48- hour treatment with YLT322 (0–20 µM) or doxorubicine (0–40 µ M). Data are expressed as the mean from three experiments.</p