Treatment with 15r (1 µM) and 15o (2 µM) on HT-29 cells caused increased production of ROS. (A) Flow cytometric analysis graphs depicting% ROS generation in HT-29 cells. (B) Intramitochondrial superoxide production was significantly enhanced in HT-29 cells after treatment with 1,2,4-triazolo-linked bis-indolyl conjugate (15a–r) derivative compounds 15r (1 µM) and 15o (2 µM), as measured by mitochondrial-localising MitoSOX Red fluorescence probe. (C) Bar representation for mean fluorescence intensity of compounds 15r and 15o. (D) Representative flow cytometry histograms of MitoSOX Red fluorescence in HT-29 cells (10,000 cells). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc analysis, where *** p < 0.001 represents control vs. 1,2,4-triazolo-linked bis-indolyl conjugates (15o and 15r) in the treatment groups.