Fig. 3: Neu2 induces apoptosis by regulating pro and anti-apoptotic molecules. | Cell Death & Disease

Fig. 3: Neu2 induces apoptosis by regulating pro and anti-apoptotic molecules.

From: Association of cytosolic sialidase Neu2 with plasma membrane enhances Fas-mediated apoptosis by impairing PI3K-Akt/mTOR-mediated pathway in pancreatic cancer cells

Fig. 3

a MIAPaCa2 cells were transfected with mock and PcDNA3.1-Neu2 expression vector and serum starved for 24 and 48 h. RNA was isolated from these cells and cDNA was prepared. RT-PCR analysis of Bax, caspase 9, and casapse3 was done. mRNA expression showed upregulation of pro-apoptotic molecules but no change was found in caspase 9 in Neu2-overexpressed MIAPaCa2 cells compared to mock. GAPDH was used as loading control. The image was visualized and photographed. b, c Real-time PCR analysis of Bax, caspase 3, and caspase 9 mRNA expressions in MIAPaCa2 and AsPC1 cells after Neu2 overexpression relative to that of mock-transfected cells after 48 h of serum starvation were determined. Values are normalized against 18S rRNA expression (n = 3 experiments). d Cell lysates were prepared from mock and Neu2-overexpressed MIAPaCa2 and AsPC1 cells and resolved by SDS-PAGE, and then analyzed by western blot with the specified antibodies. Representative immunoblots showing increased Bax, reduced level of both Bcl2 and PARP in Neu2-overexpressed PDAC cells whereas no such change was found in protein level of caspase-9. β-actin was used as loading control. e Neu2 induces apoptosis through the extrinsic-mediated apoptotic pathway. Representative immunoblots of Neu2-overexpressed MIAPaCa2 and AsPC1 cells confirmed the reduction of the protein level of Bid and enhanced caspase 8, caspase 6, Fas, FasL, and FADD which are hallmark proteins of the extrinsic apoptotic pathway. β-actin was used as loading control. f MIAPaCa2 cells were transfected with mock and PcDNA3.1-Neu2 expression vector then cells were harvested and incubated with PE-Fas (CD95) antibody for analysis by flow cytometry. Neu2 overexpressed in MIAPaCa2 cells showed upregulation of Fas. The data are the means±S.D. of three different experiments. g Neu2-overexpressed AsPC1 cells also exhibited enhanced Fas on the cell surface compared to mock-transfected cells. The data are the means±S.D. of three different experiments. Significance: *p < 0.05

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