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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: MIF/CD74 axis is a target for novel therapies in colon carcinomatosis

Fig. 6

Mitochondria impairment after 4-IPP and metformin treatments in C2 organoids. a Reactive oxygen species detection with ROS-H2O2-Glo assay kit. C2 cells were treated with metformin 5 mM, 4-IPP 100 μM or left untreated for 120 and 24 h respectively. Extracellular H2O2 formation was detected and quantified using the ROS-H2O2-Glo assay. Luminescence intensity was quantified using a microplate reader with a 500 ms integration time, reported as relative light units and normalised to 0 cells/well treatment conditions. Standard deviation was calculated for a set of triplicate values. b Confocal microscopy images of mitochondria using Mitotracker® Deep Red FM in C2 cells untreated (A2; D1; E1), treated with 50 μM 4-IPP for 24 h (B2), 100 μM 4-IPP for 24 h (C2), or 5 mM metformin for 120 h (D2; E2). Arrows indicate aggregated mitochondria. 4-IPP treatment leads to mitochondria impairment in C2 cells. Metformin treatment reduces signal intensity of Mitotracker (asterisk) suggesting a depolarization of mitochondrial membrane potential. The figure shows data from a representative experiment. DIC: differential interference contrast. Magnifications: 10X. Scale bars = 50 μm. All the experiments were replicated at least three times. C Flow cytometry analysis of the mitochondrial membrane potential using JC-1 assay on C2 organoids treated with metformin for 120 h or left untreated. Staining JC-1 revealed that metformin treatment caused a significant increase in the amount of FITC aggregates, a pattern that is characteristic for disruption of mitochondrial membrane potential. d The ultrastructure of the C2 cells after 4-IPP (D1-3) or metformin treatment (D4-6) and left untreated (D7). The white arrows show autophagic structures and red stars indicate apoptotic bodies present in 4-IPP treated cells. White triangles show irregular mitochondria, blue stars lipid droplets and the yellow star a multilamellar structure present in metformin treated cells. Additionally, no signs of autophagy, apoptosis or irregular mitochondria were observed in untreated cells (D7). e Functional consequences of 4-IPP or metformin treatments on C2 organoids

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