Activation status of Ras in pancreatic cancer cells.

<p><b>A.</b> The expression of Ras was examined by immunoblotting analysis in human pancreatic cancer BxPC-3, MIA, PANC-1 or lung epithelial BEAS-2B cells. The folds of the expression levels of Ras in pancreatic cancer cells relative to that in BEAS-2B cells were measured and indicated. Equal loading of total proteins per lane was determined by β-actin. <b>B.</b> Ras GTP-binding activity was measure in these cells by Ras-GTP assay. The blot was re-probed by anti-Ras antibody to judge evenly loading of total proteins.</p

PKC expression and activity following PMA or PMA plus GO6976 treatment.

<p><b>A.</b> Cell lysates isolated from untreated cells were immunoblotted with anti-pan-PKC antibody. <i>β</i>-actin was used to determine equal loading of total protein per lane. <b>B.</b> Cell lysates from the cells with or without being treated with PMA or PMA plus GO6976, were immunoprecipitated with anti-pan-PKC antibody. The immunocomplexes were incubated with [³²P] γ-ATP and the peptide substrates to analyze PKC activity. The error bars represented SD from three independent experiments (n = 3, <i>ρ</i><0.05).</p

Upregulation of ROS and induction of apoptosis in pancreatic cancer cells.

<p><b>A.</b> The cells were treated with GO6976 or co-treated with GO6976 plus NAC (2.5 mM). After being stained with DCF, the levels of ROS in the cells were analyzed by a flow cytometer. <b>B.</b> The cells were treated with GO6976 (1 µM) or GO6976 plus NAC. Subsequently, the samples were collected and subjected for annexin V assay. The error bars are SD from 5 independent experiments (n = 5, <i>ρ</i><0.05).</p

Induction of apoptosis in GO6976-treated pancreatic cancer cells.

<p><b>A.</b> Cells were treated with GO6976 (1 µM) for 48 h and then collected for annexin V assay. The error bars are SD from 5 independent experiments (n = 5, <i>ρ</i><0.05). <b>B.</b> Profiles of the cells after being stained with annexin V. <b>C.</b> Cells were treated with FTI for 30 min prior to GO6976 treatment. Subsequently, annexin V assay was conducted. The error bars are SD from 5 independent experiments (n = 5, <i>ρ<</i>0.05).</p

Caspase 3 activation in response to the suppression of PKC.

<p><b>A.</b> With or without GO6976 treatment, cell lysates were prepared and immunoblotted with anti-caspase 3 antibody. <b>B.</b> The cells were subjected to the treatment with GO6976 or GO6976 plus NAC. Subsequently, annexin V assay was performed. The error bars represent SD from 3 independent experiments (n = 3, <i>ρ</i><0.05).</p

Phosphorylation of p73 and upregulation of PUMA in pancreatic cancer cells.

<p><b>A.</b> With or without the treatment with GO6976 or GO6976 plus NAC, lysates were immunoprecipitated with anti-p73 antibody. The immunoprecipitates were then subjected to immunoblotting using the anti-phosphorylated serine antibody. The level of the immunoprecipitates was judged by re-probing the blot with anti-p73 antibody. <b>B.</b> Total RNAs from the cells with or without treated with GO6976, GO6976 plus NAC or GO6976 plus <i>shRNA-p73</i> infection were isolated. Equal amount of RNAs was reverse-transcribed, and the expression of <i>PUMA</i> was tested by RT-PCR. <b>C.</b> With or without GO6976 treatment, MIA or PNAC-1 cells were subjected to immunoblotting analysis for PUMA expression. Equal loading of total proteins were determined by rep-probing the blot with anti-β-actin antibody.</p

Induction of apoptosis in cultured DU145 and PC3 cells in response to PL treatment.

<p>The cells were treated with 1 mg/ml or 2 mg/ml for 48 h, and subsequently stained with Annexin V. Error bars represent the standard deviation from 3 independent experiments.</p

Analysis of side-effects of PL.

<p><b>A</b> and <b>B</b>. After the tumor nodules were formed, the body weight (<b>A</b>) and water consumption (<b>B</b>) of each mouse with or without treated with PL were measured. Error bars represent the standard deviation from 3–6 mice.</p

Attenuation of the tumors formed by the inoculation of DU145 or PC3 cells into nude mice after the treatment with PL.

<p><b>A</b>. DU145 or PC3 cells were inoculated subcutaneously into nude mice. Twelve days later, the photos of the mice bearing the inoculated tumors were taken (upper panel). The tumors were isolated from PL treated or control mice. The slides mounted with the tumor tissues were prepared and stained with HE dye (lower panel). <b>B</b> and <b>C</b>. A group of 6 mice were injected with PL (30 mg/kg) in water from day 0 and subsequently administrated every 2 days. Another group of 3 was as the controls. One week later when the inoculated tumor started to form, the sizes of the tumors were measured every 4 days.</p

Histopathology or immunohistochemistry staining of PL-treated or control tumors isolated from the mice.

<p><b>A</b>. The tumors were prepared for hematoxylin and eosin staining. <b>B</b> and <b>C</b>. The slides mounted with the tumor tissues were stained with TUNEL agent (<b>B</b>) or anti-caspase 3 antibody (<b>C</b>).</p