Fig 2: Loss of condensin II CAP protein expression results in aberrant mitochondrial respiratory function. Seahorse mitochondrial stress test assays were performed in (A) NT (n=21) and NCAPD3 (n=21, n=18) shRNA-expressing (three experimental replicates), (B) NT (n=20) and NCAPH2 (n=20) shRNA-expressing (two experimental replicates), and (C) control (n=30) and NCAPH2 (n=30) siRNA-transfected (three experimental replicates) cells. Cells were injected with drugs at the time points indicated: (A) 1 µM oligomycin; (B) 0.6 µM FCCP; (C) 1 µM antimycin A and rotenone. The first graph in each panel illustrates the full OCR profile, showing OCRs (y-axis) over time (x-axis). The second graph shows mitochondrial basal respiration rates. This value is calculated by subtracting non-mitochondrial respiration rates (OCR values post treatment with antimycin A and rotenone) from cellular respiration rates measured prior to addition of any mitochondrial inhibitory compounds. The final graph shows maximal (uncoupled) respiration rates. This value is measured after the addition of FCCP, a compound that uncouples oxygen consumption from ATP production. All values are normalized to DNA content by measuring the absorbance value at 485 nm relative to that at 535 nm using the Cyquant Cell Proliferation assay to account for possible differences in cell number. *P≤0.05. (Student's t-test). (D) Western blot analysis of NCAPH2 levels in control and NCAPH2 siRNA-transfected cells. β-tubulin served as a loading control.

Fig 3: NCAPD3-depleted cancer cells exhibit mitochondrial complex I-III dysfunction. High-resolution respirometry was performed to measure OCRs in intact non-permeabilized HT-29 cells in mitochondrial respiration buffer, using the Oroboros instrument. Cells were permeabilized with digitonin and ETC complex-specific substrates and inhibitors were added, sequentially, as described in the Materials and Methods (Kumar et al., 2019; Pesta and Gnaiger, 2012). Proton leak and oxidative phosphorylation (OXPHOS) in response to complex I–III substrates malate (M), pyruvate (P), ADP (D), glutamate (G) and complex II substrate, succinate (S) were measured. complex II activity was determined by subtracting the OCRs following addition of malate, pyruvate and glutamate from the OCRs following addition of malate, pyruvate and glutamate and succinate. Maximum respiration (Max R) as well as reserve respiratory (RR) capacity (response to the protonophore FCCP) were quantified. Rotenone-sensitive and -insensitive respiration, followed by complex IV function were also measured. Experiments were performed in HT-29 cells induced to express (A) NCAPD3 shRNA 1 (red bars) or (B) NCAPD3 shRNA 2 (gray bars) and results were compared to HT-29 cells expressing NT shRNA (blue bars). Four biological replicates were performed for each cell line. *P<0.05, **P<0.005; NS, not significant (paired t-tests).

Fig 4: NCAPD3 and NCAPH2 affect the expression of a few mitochondria-associated genes in a similar manner. (A) Venn diagram comparing the shared deregulated mitochondrial associated genes in NCAPD3 (red circle) and NCAPH2 (green circle)-depleted cells. (B) Table detailing the shared deregulated mitochondrial associated genes, fold change and gene function.

Fig 5: NCAPD3 and NCAPH2 localize to different mitochondrial subcompartments. (A) Diagram of mitochondria and localization patterns of mitochondrial proteins. (B) Equal amounts of mitochondrial lysate with or without proteinase K treatment were immunoblotted for NCAPD3. Mfn1 served as an outer membrane control, CoV served as an inner membrane control, and ALDH2 served as a matrix control. NCAPD3 levels were quantified in the lysates and normalized to the level of CoV. n=3 independent experimental replicates. (C) Equal amounts of mitochondrial lysate with or without proteinase K treatment were immunoblotted for NCAPH2. Mfn1 served as an outer membrane control, CoV served as an inner membrane control. NCAPH2 levels were quantified in the lysates and normalized to the level of CoV. n=3 independent experimental replicates. (D) Equal amounts of mitochondrial lysate with or without proteinase K treatment were immunoblotted for SMC2. Mfn1 served as an outer membrane control and CoV served as an inner membrane control. SMC2 levels were quantified in the lysates and normalized to CoV. n=2 independent experimental replicates. *P≤0.05; NS, not significant (Student's t-test).