Fig 1: Phycocyanin blocks G2/M cell cycle progression and induces caspase 3 independent cell death in PANC-1 cells.(A) The effect of phycocyanin on the cell cycle distribution of PANC-1 cells. PANC-1 cells treated with indicated concentrations of phycocyanin for 72 h were fixed, stained and analyzed for DNA content by FACS. (B) The distribution and percentage of cells in G0/G1, S and G2/M phase of the cell cycle were calculated and plotted. The statistically significant differences between the treated cells and the control group were indicated by *P < 0.05, **P < 0.01. (C) Nuclear shrinkage ratio was analyzed with cell scoring model of High Content Screening system software. The data shown were representative of three independent experiments. The statistically significant differences between the treated cells and the control group were indicated by *P < 0.05, **P < 0.01. (D) Flow cytometry analysis of PANC-1 cell apoptosis induced by phycocyanin for 72 h. (E) Following treatment with phycocyanin, early apoptotic cell population with Annexin V-positive and late apoptotic cell population with PI- positive PANC-1 cells increased in a dose-dependent manner. Mean ± SD of three assays. *P < 0.05, **P < 0.01, versus control. (F) Knockdown of caspase 3 by siRNA pool for 24 h followed by 10 µM phycocyanin treatment for 48 h decreases capase 3 expression in PANC-1 cells when compared to cells treated with lipofectamine alone (NS) or non-silencing siRNA (Control siRNA) and 10 µM phycocyanin. (G) Caspase 3 siRNA inhibited the activation of caspase 3 in PANC-1 cells induced by phycocyanin. Knockdown of caspase 3 by siRNA pool for 24 h followed by 10 µM phycocyanin treatment for 72 h decreases caspase 3 activation. The bars represent mean ± SD, n = 3, **P < 0.01, NA: No significance (t- test). (H) Dose-dependent growth cell inhibition by phycocyaninon in PANC-1 cells transfected with caspase 3 or control siRNA. PANC-1 cells transfected with caspase 3 or control siRNA were treated with indicated concentrations of phycocyanin for 48 h. Data were means ± SD of three independent experiments. *P < 0.05, **P < 0.01 (t-test).
Fig 2: Effect of phycocyanin on cell viability and apoptosis marker expression in PANC-1 cells in the case that the MAPK and the NF-?B pathway are separately and simultaneously suppressed.(A) The nuclear localization of NF-?B after phycocyanin treatment was blocked by SN50. Cells were treated with 10 µM phycocyanin for different time with SN50 (10 µM). Cytosolic and nuclear proteins were used for western blot analysis using anti-NF-?B p65 antibody. (B) Effect of phycocyanin on PANC-1 cell viability treated with SN50. The data shown are representative of three independent experiments. The bars represented mean ± SD, n = 3. *P < 0.05, **P < 0.01, NA: No significance (t-test). (C) Western blot analysis of nucleus NF-?B, LC3 II/I, Beclin 1, procaspase, caspase and PARP expression in PANC-1 cells treated with 10 µM phycocyanin in absence or presence of SN50 (10 µM) for indicated time point. (D) Activation of p38 was effectively inhibited by the specific MEK inhibitor PD98059. (E) Activation of JNK was effectively inhibited by the specific MEK inhibitor PD98059. (F) Effect of phycocyanin on PANC-1 cell viability treated with PD98059. The data shown are representative of three independent experiments. The bars represented mean ± SD, n = 3, *P < 0.05, **P < 0.01, NA: No significance (t-test). (G) Western blot analysis of procaspase, caspase 3 and PARP expression in PANC-1 cells treated with 10 µM phycocyanin in absence or presence of PD98059 (5 µM) for 48 h. (H) Effect of phycocyanin on PANC-1 cell viability after inhibition of MAPK pathway alone and simultaneous inhibition of MAPK and NF-?B pathway. The bars represent mean ± SD, n = 3. Control group (NS) vs PD98059 group: *P < 0.05, **P < 0.01(t test); “PD98059” group vs “PD98059 + SN50” group: #P < 0.05, ##P < 0.01 (t-test).
Fig 3: Inhibition of autophagy by Beclin 1 siRNA rescues phycocyanin-mediated cell death in PANC-1 cells.(A) Knockdown of Beclin 1 by siRNA pool for 24 h followed by 10 µM phycocyanin treatment for 48 h decreases Beclin 1 expression in PANC-1 cells when compared to cells treated with non-silencing siRNA (Control siRNA) and 10 µM phycocyanin. (B) Treatment of PANC-1 with 10 µM phycocyanin for 24 h following a knockdown of Beclin 1 gene with siRNA pool for 48 h shows a significant decrease in MAP-LC3 punctate pattern as compared to cells treated with non-silencing siRNA (control siRNA) or lipofectamine alone followed by phycocyanin. (C) Quantification of the percentage of cells with focal MAP-LC3 at the indicated conditions. Error bars correspond to SD of three repeated wells, counting 200 cells each. *P < 0.05; **P < 0.01, 0 h versus phycocyanin-treated cells. (D) PANC-1 cells treated with 10 µM phycocyanin for 48 h following Beclin 1 only and Beclin1 and caspase 3 dual silencing showed a significant decrease of cell viability inhibition when compared to cells treated with control siRNA alone along with phycocyanin. The bars represent mean ± SD, n = 3, **control siRNA group vs Beclin 1 siRNA group, P < 0.01; ##“Beclin 1 siRNA” group vs “Beclin 1 siRNA + Caspase 3 siRNA” group, P < 0.01 (t test).
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