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. 2022 Aug 31;23(17):9902. doi: 10.3390/ijms23179902

Figure 3.

Figure 3

HCA enhances Caspase-3/-7 activity and induces apoptosis in pancreatic cancer cells mainly through the extrinsic pathway. (A) Caspase 3/7 activity was measured in MIA PaCa-2 cells as a luminescence read-out (Caspase-3/7 Glo assay) after incubation with HCA (200 µM for 48 h: the bars correspond to the mean ± SEM values of 2 independent experiments performed in triplicate). (B) Representative immunoblot of the effects of C8i (inhibitor zIETD-fmk, 50 µM), C9i (inhibitor zLEHD-fmk, 50 µM), and C8i+C9i (zIETD-fmk+zLEHD-fmk, 50 + 50 µM) on PARP proteolysis in MIA PaCa-2 cells treated with HCA for 48 h. MIA PaCa-2 cells were pretreated with the inhibitors for 1.5 h before exposure to HCA (200 µM). (C) Effects of C8i, C9i, and C8i+C9i on the survival of MIA PaCa-2 cells treated for 48 h with HCA (the bars correspond to the mean ± SEM of three independent experiments performed in triplicate). (D) Labelling of the Fas receptor (green) and Wheat germ agglutinin (red) in MIA PaCa-2 cells cultured for 48 h in the presence or absence of HCA (150 and 200 µM). Capping of the Fas receptors is indicated with arrows. Scale bar 4 µM. Student’s t-test: ** p < 0.01, *** p < 0.001 with respect to the controls. HCA, 2-hydroxycervonic acid; C8i, caspase 8 inhibitor (zIETD-fmk); C9i, caspase 9 inhibitor (zLEHD-fmk); C8i+C9i, caspase 8 inhibitor + caspase 9 inhibitor (zIETD-fmk+zLEHD-fmk); PARP, Poly (ADP-Ribose) Polymerase; WGA, Wheat germ agglutinin; –, the absence of the drug indicated in the figure.