Bcl-xL is up-regulated in liver tumor tissues and HCC cells.

<p><b>A</b>. Lysates from normal liver tissues and liver tumor tissues of different patients were prepared and subjected to Western blotting. Bcl-xL, Mcl-1, and survivin expressions were detected with specific antibodies. β-actin protein levels were assessed as an equal protein loading control (P1, P2, and P3: patient number). <b>B</b>. Cell lysates from human normal primary hepatocytes and HCC cells LH86 and Huh7 were prepared and Western blotting was performed to detect Bcl-xL, Mcl-1, and survivin expression with specific antibodies. β-actin protein levels were assessed as an equal protein loading control.</p

ABT-263 induces activation of ERK and survivin up-regulation in HCC cells.

<p><b>A.</b> LH86 cells were treated with different doses of ABT-263 as indicated for 1 h. Cells were harvested and cell lysates were prepared for Western blotting. Phospho-ERK and ERK protein levels were examined with specific antibodies. β-actin was assessed and served as an equal protein loading control. <b>B</b>, LH86 cells were untreated or treated with ABT-263 (1 µM) for 1 h. Cells were harvested and cell lysates were prepared for Western blotting. Survivin expression level was examined with specific antibodies. β-actin was assessed and served as an equal protein loading control. <b>C.</b> LH86 cells were not treated (control) or treated with ABT-263(1 µM), ERK specific inhibitor PD98059 (50 µM), or pre-treated with PD98059 (50 µM) for 1 h followed by ABT-263 (1 µM) for 24 h. Apoptosis was determined by Hoechst staining to show cells with apoptotic nuclei (representative apoptotic cells were labeled with white arrows; PD: PD98059). <b>D.</b> LH86 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g006" target="_blank">Figure 6C</a>, apoptosis was assessed by nuclear staining and cells with apoptotic nuclei were counted as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#s2" target="_blank">materials and methods</a>’ (*p<0.05). <b>E.</b> LH86 cells were untransfected or transiently transfected with synthesized random siRNA (control) or ERK specific siRNA duplexes, and 48 h post-transfection, cells were subjected to Western blotting analysis with anti-ERK polyclonal antibody. β-actin was used as an equal protein loading control. <b>F.</b> LH86 cells were transfected with synthesized random control siRNA or ERK specific siRNA, and 48 h post-transfection, cells were untreated or treated with ABT-263 (1 µM) for 24 h and then subjected to Hoechst staining to show apoptotic cells with condensed nuclei (representative apoptotic cells were marked with white arrows).</p

Survivin down-regulation sensitizes ABT-263-induced apoptosis in HCC cells.

<p><b>A.</b> LH86 cells were treated as indicated and cell lysates were prepared for Western blotting. Pro-apoptotic proteins: Bax, Bad, and Bak and anti-apoptotic proteins Bcl-xL and Mcl-1 were assessed with specific antibodies respectively. β-actin was detected and served as an equal protein loading control. <b>B.</b> LH86 cells were untreated or treated with ABT-263 (1 µM), YM-155 (1 µM) or combination of ABT-263 (1 µM) and YM155 (1 µM) for up to 6 h as indicated. Then cells were harvested and cell lysates were prepared for Western blotting. Anti-survivin and anti-Bcl-xL polyclonal antibodies were used to assess protein levels for survivin and Bcl-xL respectively. β-actin was used as an equal protein loading control. The band intensities of survivin, Bcl-xL, and β-actin was qualified with Image J software. <b>C.</b> LH86 cells were transiently transfected with synthesized random siRNA (control) or survivin specific siRNA duplexes, and 48 h post-transfection, cells were subjected to Western blotting analysis with anti-survivin polyclonal antibody. β-actin was used as an equal protein loading control. <b>D.</b> LH86 cells were transfected with synthesized random control siRNA or survivin specific siRNA, and 48 h post-transfection, cells were untreated or treated with ABT-263 (1 µM) for 24 h and then subjected to Hoechst staining to show apoptotic cells with condensed nuclei (representative apoptotic cells were marked with white arrows). <b>E.</b> LH86 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g004" target="_blank">Figure 4D</a> and apoptosis was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g002" target="_blank">Figure 2A</a>. Statistical analysis was performed for apoptosis ratio by counting the number of cells with apoptotic nuclei (*p<0.05). <b>F.</b> LH86 cells treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g004" target="_blank">Figure 4D</a> were harvested and cell lysates were prepared and subjected to Western blotting. Apoptosis was determined through caspase 3 activation. β-actin was used as an equal protein loading control.</p

Bcl-xL down-regulation enhances YM-155-induced apoptotic toxicity in HCC cells.

<p><b>A.</b> Huh7 cells were transiently transfected with synthesized random siRNA (control) or Bcl-xL specific siRNA duplexes, and 48 h post-transfection, cells were subjected to Western blotting analysis with anti-Bcl-xL polyclonal antibody. β-actin was used as an equal protein loading control. <b>B.</b> Huh7 cells were transfected with synthesized random control siRNA or Bcl-xL specific siRNA, and 48 h post-transfection, cells were untreated or treated with YM-155 (1 µM) for 24 h and then subjected to Hoechst staining to show apoptotic cells with condensed nuclei (representative apoptotic cells were marked with white arrows). <b>C.</b> Huh7 cells were treated as in with YM-155 as indicated and apoptosis was measured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g002" target="_blank">Figure 2A</a>. Statistical analysis was performed for apoptosis ratio by counting the number of cells with apoptotic nuclei (*p<0.05). <b>D.</b> Huh7 cells treated were harvested and cell lyates were prepared and subjected to Western blotting. Apoptosis was determined through caspase 3 activation. β-actin was used as an equal protein loading control.</p

YM-155 sensitizes ABT-263-induced apoptosis in HCC cells.

<p><b>A.</b> LH86 and <b>B.</b> Huh7 cells were untreated or treated with ABT-263(1 µM), YM-155(1 µM) or combination of ABT-263(1 µM) and YM-155(1 µM) for up to 6 h. Then apoptotic cells were assessed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#pone-0021980-g002" target="_blank">Figure 2A and 2B</a> (representative apoptotic cells were marked with white arrows). <b>C.</b> LH86 and <b>D.</b> Huh7 cells were untreated or treated with ABT-263(1 µM), YM-155(1 µM) or combination of ABT-263(1 µM) and YM-155(1 µM) for 6 h. Cells with apoptotic nuclei were counted to determine cell death ratio (*p<0.05, **p<0.05). <b>E.</b> LH86 cells and <b>F.</b> Huh7 cells were treated as indicated and cell lysates were prepared and subjected to Western blotting. Apoptosis was evaluated through caspase 3 activation. β-actin was used as an equal protein loading control. <b>G.</b> LH86 cells grown in six-well plate were untreated (control) or treated with different conditions as indicated for 48 h. After rinsed with fresh culture medium for 3 times, cells were cultured for another two weeks. Cell colony formation assays were performed with crystal violet staining. <b>H.</b> colony number were counted to show combination treatment with ABT-263 and YM-155 resulted in reduction of clonogenesis (#p<0.05).</p

HCC cells are resistant to low doses of ABT-263.

<p><b>A.</b> LH86 and <b>B.</b> Huh7 cells were treated with ABT-263 (0–20 µM) for up to 24 h. Apoptosis was measured through Hoechst staining to show apoptotic cells with condensed nuclei as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021980#s2" target="_blank">materials and methods</a>’. (representative apoptotic cells were marked with white arrows in ABT-263 treatment panel). <b>C.</b> HCC cells were treated with increasing doses of ABT-263 as indicated for up to 24 h. Then cells were harvested and cell lysates were prepared and subjected to Western blotting. Caspase activation was assessed through detecting the cleaved bands of caspase 9 and caspase 3. β-actin protein levels were used as an equal protein loading control.</p

Characterization of aptamer TLS11a.

<p>(a) The secondary structure of aptamer TLS11a and its binding ability to (b) LH86 and (c) human normal liver cells, Hu1082. The green histogram shows the background binding (control aptamer, TD05), and the red fluorescence intensities show the binding of TLS11a with target and control cells. All probes were labeled with Phycoerythrin-Cy5.5. (d) Kd determination.</p

Relative cell viability and apoptosis of cells treated with TLS11a-GC, TD05-GC, free Doxorubicin, TLS11a-GC-Dox or TD05-GC-Dox.

<p>(a) Relative cell viability of LH86 (target cell line); (b) Relative cell viability of Hu1229 (human normal liver cells); (c) Hoechst 33258 staining for apoptotic and dead LH86 cells treated with a series of concentrations of TLS11a-GC-Dox or TD05-GC-Dox; (d) Western blot results for cleaved caspase 3 in LH86 cells treated with a series of concentrations of TLS11a-GC-Dox or TD05-GC-Dox.</p

Tumor inhibition of the aptamer-Dox complex in mouse model.

<p>(a) Average tumor volume of mice treated with nothing (black), Doxorubicin (red), or TLS11a-GC-Dox (blue); (b) Microscopy images of H&E stained tumor tissue slides.</p