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. 2021 Mar 13;6(10):3300–3313. doi: 10.1016/j.bioactmat.2021.03.005

Fig. 5.

Fig. 5

In vitro apoptosis evaluation and mechanisms assay. (A) Annexin V-FITC/PI apoptotic staining for MCF-7 ADR cells treated with NapFFGEE-JSK hydrogel or alkynyl-JSK prodrug (62 μM) at 48 h was evaluated by flow cytometry analysis, the percentages of early or late apoptosis are presented in the bottom right and top right quadrants, respectively. And right columns represent the average proportions of apoptotic cells. (*p < 0.05, **p < 0.01, ***p < 0.001; n = 3). (B) Intracellular oxidative stress-elevation of NapFFGEE-JSK hydrogel or alkynyl-JSK treatment at 48 h via GSH/GSSG ratio and ROS levels assays. (C) Mitochondrial dysfunction induced by oxidative stress from NapFFGEE-JSK or alkynyl-JSK treatment for 48 h was accessed by the analysis of the mitochondrial membrane potential and ATP production. (D) Western blotting for the apoptosis proteins expression of Bcl-2, Bax, Cytochrome c, Cleaved Caspase 9 and Cleaved Caspase 3. The levels of protein expression were detected using specific antibodies and assayed by Image J software. The data are expressed as Mean ± SDs (n = 3). *p < 0.05; **p < 0.01, ***p < 0.001. (E) Schematic illustration of the mitochondrion mediated apoptotic mechanism of NapFFGEE-JSK supramolecular hydrogel on MCF-7 ADR cells.