Fig 1: Cellular bioenergetics analysis of hAFSCs from normal and fetus-affected gestations. (A) Assessment of cellular energy flux for hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations, shown as a percentage relative to hAFSCs from fetus-unaffected gestations. Comparative measurements were taken using Abcam Extracellular oxygen consumption assay (ab197243) and Abcam Glycolysis assay (ab197244). (B) Analysis of mitochondrial membrane potential (??m) of hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations; values are indicated as mean ± SD. Analysis was performed using Abcam TMRE Mitochondrial membrane potential assay kit (ab113852). (C) Assessment of cellular energy content (ATP) of hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations. Measurements were performed using Abcam Luminescent ATP detection assay kit (ab113849). (D–F) ROS production measurement in hAFSCs, obtained from fetus-unaffected (Normal, n = 3) and fetus-affected (Pathology, n = 3) gestations, performed using Abcam DCFDA Cellular ROS detection assay kit (ab113851). Qualitative evaluation with fluorescence microscopy (D): representative images of ROS production in Normal and Pathology groups (scale bar = 400 µm). Quantitative evaluation with flow cytometry (E,F) representative images of ROS production (E) and median fluorescence intensity evaluation (F) in Normal and Pathology [Pat2] groups. (G) RT-qPCR analysis of genes related to cell metabolism and respiration: HIF1A, NRF1, PPARGC1A, ERRA, PKM, LDHA, PDK1, CAT1, SOD2, and GPX1 in control hAFSCs from normal and fetus-affected gestations. mRNA expression levels were normalized to GAPDH and RPL13A and presented as mean values of ?Ct ± SD. *Denotes significant difference with p < 0.05, as evaluated using Student’s t-test.
Fig 2: Effect of metformin and MCL-1 inhibitor S63845 on myeloid leukemia cell mitochondrial membrane potential and cellular reactive oxygen species production. For subsequent flow cytometric analysis NB4 cells were treated with 25 nM and 50 nM S63845, 10 mM metformin and combination of 25 nM S63845 + 10 mM metformin. KG1 cells were treated with 100 nM and 250 nM S63845, 10 mM metformin and combination of 100 nM S63845 + 10 mM metformin. KG1A cells were treated with 1000 nM and 2000 nM S63845, 10 mM metformin and combination of 1000 nM S63845 + 10 mM metformin. (a) Mitochondrial membrane potential (??m) of control (untreated) and treated NB4, KG1 and KG1A cells was evaluated using TMRE Mitochondrial membrane potential assay kit (ab113852) after 24 and 72 h of incubation. (b) Measurements of ROS production in control (untreated) and treated NB4, KG1 and KG1A cells were performed using Abcam DCFDA Cellular ROS detection assay kit (ab113851) after 24 and 72 h of incubation. Note: * denotes significant difference between treated vs. control cells with p < 0.05, ** denotes significant difference with p < 0.01, *** denotes significant difference with p < 0.005 and **** denotes significant difference with p < 0.0001, as evaluated using 1-way ANOVA with Dunnett post hoc test.
Supplier Page from Abcam for TMRE-Mitochondrial Membrane Potential Assay Kit