Fig. 3: TM treatment impacts mitochondrial Complex IV.

a Western blot showing respiratory complexes in LM2 cells following 72-h TM treatment (0, 0.1, 0.2, 0.5 µM). Complexes I–V are referred to as CI (mol. wt. ~19 kDa), CII (mol. wt. ~30 kDa), CIII (mol. wt. ~48 kDa), CIV (mol. wt. ~40 kDa), and CV (mol.wt. ~55 kDa), respectively. NDUFA4 is a subunit of Complex IV. b qPCR analysis showing transcript levels of human Complex IV subunit 4I1 (COX4I1) in control and TM (0.5 µM) treated LM2 cells, treated with TM for 72 h. Analysis was performed by unpaired two-sided t-test. p-value = 0.079. c Protein expression of cytochrome c with and without TM treatment (0.5 µM) (n = 4/group). d Human cytochrome c oxidase activity (Abcam) kinetically measured over 2 h after 72 h of treatment with TM (0.5 µM) in LM2 cells. The graph represents the difference in Complex IV activity at 110th minute during measurement. Analysis was performed by unpaired two-sided t-test (n = 4/group). p-value = 0.0063. Center lines of box plots denote median values, top whiskers denote maxima and bottom whiskers minima. e Mitochondrial DNA content in LM2 cells as determined by qPCR (p = 0.060). Analysis was performed by unpaired two-sided t-test (n = 4/group). f–h MitoTracker deep red analysis in control and TM (0.5 µM) treated LM2 (f), ML1 (g) and MDA-MB-468 (h) cells, treated for 72 h (n = 3/group). i Structure of cristae in the inner mitochondrial membrane as determined by transmission electron microscopy (×50,000), red arrows indicate normal/folded and perturbed cristae morphology in LM2 cells. Scale bar, 500 nm. j Quantification of perturbed cristae per 100 units of mitochondrial area in pixels (n = 30 mitochondria in controls, n = 36 mitochondria for TM group). Analysis was performed using Mann–Whitney two-sided test. Representative data of two independent experiments are depicted. Results are expressed as mean ± SD. **p < 0.01 ****p < 0.0001.