Figure 3
bLf-Dox conjugates treatment induces cancer cell apoptosis.
(A) Representative confocal microscopy images of DU145 cells showing the expression of annexin-V as a marker of apoptosis post treatment with Dox, Apo-bLf-Dox and Fe-bLf-Dox. The annexin-V was stained using immunofluorescence with rabbit anti-annexin-V primary antibody and anti-rabbit-IgG FITC conjugated secondary antibody represented in green fluorescence. Dox auto-fluorescence is represented in red. The nucleus was counterstained with DAPI (blue). Scale bar = 50 μm. (B) Percentage of cellular annexin-V expression from 5 images each was calculated and represented as a histogram (Mean ± S.D.). (C) The presence of fragmented DNA as an end product of apoptotic cascade within the cell was considered as the confirmatory test for induction of apoptosis especially the Dox mediated DNA damage using TUNEL assay. The nucleus was counterstained with DAPI and Dox auto-fluorescence is represented in red. (D) Percentage of TUNEL positive cells from 5 images each has been represented as a histogram. Statistical analysis was performed using one-way ANOVA and a post-hoc Tukey’s test. (E) The molecular regulation of apoptosis was studied using Western blots for various apoptotic markers. The Western blotting was carried out with 100 μg of complete cell lysates against respective primary and secondary antibodies. (F) The Western blot images acquired were analysed for band density using ImageJ (NIH) software and the protein expression was given in relative fold change as compared to the untreated sample normalised against GAPDH and expressed as a histogram.
