Fig. 3

Mammospheres exhibit fragmented mitochondrial morphology and are metabolically less active. a Western blot analyses were performed to assess the protein levels of mitochondrial fission markers (DRP1, pDRP1, and FIS1, indicated in green), mitochondrial fusion markers (OPA1 and MFN1, indicated in blue), and ETC complex proteins (NDUFA9, UQCRC2, and OSCP, indicated in red) in patient breast tumors compared to adjacent normal tissues (n = 10) and in MDA-MB-231 mammospheres in comparison with the adherent cell population (n = 3). b Comparison of OPA1 oligomerization pattern between MDA-MB-231 mammospheres and adherent cell population (n = 3). c Representative confocal microscopic images analysing the mitochondrial morphology of MDA-MB-231 adherent and mammosphere cultures as observed using indirect immunofluorescence for TOM20 (mitochondria, red; nuclei stained with DAPI, cyan). Scale bar: 20 µm (n = 3). Quantification of d length and e connectivity of mitochondria in mammospheres and adherent cell population (n = 3; Each point represents length or connectivity of individual cells). f Bar plots representing quantification of mitochondrial morphology in mammospheres in comparison to adherent cell population (n = 3). g The bar graph quantifies TMRM signals in MDA-MB-231 cells and mammospheres (n = 3). h Quantification of ATP levels and i lactate levels in MDA-MB-231 cells and mammospheres (n = 4). j Mitochondrial respiration rate as reflected by the oxygen consumption rate (OCR) was analysed using Seahorse XF-24 analyzer. The evaluation was performed on MDA-MB-231 adherent cells and mammospheres under basal conditions and following the addition of oligomycin (2 μM), FCCP (1 μM), or rotenone/antimycin (0.5 μM). OCR levels were normalized to the concentration of total protein (n = 3). k Bar plots representing basal respiration rate and l ATP-linked OCR in mammospheres when compared to adherent cell population (n = 3). m Percentage of non-viable cells were measured using trypan blue exclusion assay in MDA-MB-231 adherent cells and mammospheres after treatment with OXPHOS inhibitors: rotenone, antimycin, CCCP and oligomycin (n = 3). n Relative cell viability was measured using MTT assay in MDA-MB-231 adherent cells and mammospheres after treatment with OXPHOS inhibitors: rotenone, antimycin, CCCP and oligomycin (n = 3). All protein expressions were normalized against β-tubulin, which served as the internal loading control. The data are presented as the mean ± standard deviation (SD), with “n” representing the number of biological replicates per experimental group. Significance was assessed using two-way ANOVA followed by Tukey’s post hoc test for Fig. 3f and unpaired Student’s t-test for rest of the experiments. The associated p-values are indicated in the bar plots. Compared to their respective control group: *p < 0.05, **p < 0.01, and ***p < 0.001. N Normal, T Tumor, 231 MDA-MB-231, 2D Adherent cells, 3D Mammospheres, β-TUB β-tubulin, BMH 1,6-Bis(maleimido)hexane, mt Mitochondria. Adherent cells; 3D Mammospheres, OCR Oxygen consumption rate, O oligomycin, F FCCP, R/A rotenone/antimycin