The pathway of circadian rhythm in radish.

<p>The genes in colored rectangular box are DEGs in RT vs. VE, and that in colored rhombic box are DEGs in VE vs. VL. The up-regulated genes are in red and the down-regulated genes are in green.</p

Global analysis of transcriptome datasets of the nine samples.

<p>The y-axis indicates the percentage of expressed transcripts after filtering.</p

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

Venn diagram of numbers of DEGs between different treatments.

<p>Venn diagram of numbers of DEGs between different treatments.</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

qRT-PCR verification of differentially expressed genes.

<p>Gene expression differences analyzed by qRT-PCR and RNA-seq are both exhibited. For qRT-PCR, error bars indicate the standard deviation (STDEV) for the replicates in each experiment.</p

The correlation coefficients (<i>R</i>^<i>2</i>) among the samples.

<p>The correlation coefficients (<i>R</i>^<i>2</i>) among the samples.</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

Hierarchical clustering graph of genes with expression difference in VE vs. VL but not in RT vs. VE.

<p>Hierarchical clustering graph of genes with expression difference in VE vs. VL but not in RT vs. VE.</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