Fig 1: TCR stimulation induces a robust functional reprogramming of T-cell mitochondria.(A, B) Resting and maximal mitochondrial oxygen consumption rates (OCR), and extracellular acidification rates (ECAR) in effector CD4+ T-cells from 3 donors measured using the Seahorse assay. (B) Quantified mean ± SEM of 3 healthy donors/biological replicates. *p ≤ 0.05 (Basal p = 0.0471; Maximal p = 0.0275; ECAR p = 0.0274), assessed by two-tailed paired Student’s t-test. (C) ATP levels (assessed by the Cell Titer Glo® Luminescence Assay) upon CD4+ T-cell activation. Quantification shows mean ± SEM of 6 healthy donors/biological replicates. **p < 0.01 (p = 0.0083), assessed by two-tailed paired Student’s t-test. (D) Mitochondrial H2O2 levels in naive and effector CD4+ T-cells. Quantifications show a mean ± SD of 43 naive and 71 effector T-cells/biological replicates from 3 healthy donors. ****p < 0.0001 (p = 8.9134E−07), assessed by two-tailed unpaired Student’s t-test. (E) Single cell measurements of ∆Ψm using TMRE in naive and effector T-cells. Traces show mean RFU of 782 naive and 678 effector T-cells/biological replicates from 4 healthy donors. The quantification on the right shows mean ± SD of the resting ∆Ψm. CCCP: 2 μM. ****p < 0.0001 (p = 0), assessed by two-tailed unpaired Student’s t-test. RFU = relative fluorescence units. (F) Single cell measurements of ∆Ψm using JC-1 staining. Traces and quantification show mean ± SD of 121 naive and 164 effector T-cells/biological replicates from 2 healthy donors. CCCP: 2 μM. ****p < 0.0001 (p = 1.41716E−40), assessed by two-tailed unpaired Student’s t-test. (G) Mitochondrial protein translation measured at 0, 24, 48, and 72 hrs after TCR activation, using metabolic 35S labelling. The blot is representative of one donor and the quantification is a mean ± SEM of 3 different donors/biological replicates (single points), as percentage of ‘Naive control’. ***p < 0.001; *p ≤ 0.05 (COX1 p = 0.0278; CYTB p = 0.0138; ND2 p = 0.0484; ND1 p = 0.0384; COX2/COX3 p = 0.0004; ATP6 p = 0.0065; ATP8 p = 0.0428), assessed by two-tailed paired Student’s t-test. E = Effector. Source data are available online for this figure.
Fig 2: COX Vmax in iPSC models. (A) COX Vmax measured in ND and AD iPSC derived neurons; data are normalized to ND. (B) COX Vmax among female and male groups of ND and AD iPSC derived neurons, data are normalized to ND. (C) COX Vmax measured in ND and AD iPSC derived astrocytes; data are normalized to ND. (D) COX Vmax among female and male groups of ND and AD iPSC derived astrocytes, data are normalized to ND. (E) COX Vmax measured in ND and AD iPSC derived cerebral organoids; data are normalized to ND. (F) COX Vmax among female and male groups of ND and AD iPSC derived cerebral organoids, data are normalized to ND. ND, non-demented; AD, Sporadic Alzheimer’s disease. * Indicates p ≤ 0.05, *** indicates p ≤ 0.001, **** indicates p ≤ 0.0001, and ns indicates non-significant.
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.
Fig 4: IMS-localized FKBP4 plays a critical role in controlling mitochondrial respiration in COAD cells.(A) Immunoblotting for the indicated proteins in cytosolic, nuclear, and mitochondrial (Mito) fractions of HCT116 cells transfected with an empty vector (EV) or 6xHis-tagged FKBP4 fused with mitochondrial-targeting signal from SMAC (MTS1-FKBP4) followed by treatment with siCtrl or siRNA targeting 3′ UTR of the endogenously transcribed FKBP4 mRNA (siFKBP4_3′UTR). (B) Proteinase K protection assay of mitochondria isolated from HCT116 cells that were transduced with MTS1-FKBP4 and transfected with siFKBP4_3′UTR. PK, proteinase K. MM, mitochondrial matrix; IMS, intermembrane space; OMM, outer mitochondrial membrane. TFAM (MM protein), CYCS (IMS protein), and Tom20 (OMM protein) were used as the control showing the extent of digestion. (C, D) Profiles of mitochondrial respiration over time in HCT116 cells infected with an empty vector (EV) or MTS1-FKBP4 followed by the transfection of siCtrl or siRNA targeting 3′UTR of endogenous FKBP4 mRNA (siFKBP4). O, oligomycin; F, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP); R+A, rotenone and antimycin A. (D) Basal, ATP-linked respiration, and maximal respiratory capacity were calculated. ***P < 0.001; ns, not significant by the two-tailed t test. Data are presented as mean + SD. n = 6. (E) Western blot analysis of the indicated proteins in cytosolic, nuclear, and mitochondrial (Mito) fractions of HCT116 cells that were infected with an empty vector (EV) or 6xHis-tagged FKBP4 fused with mitochondrial targeting signal from COX4A (MTS2-FKBP4) followed by treatment with siCtrl or siFKBP4_3′UTR. (F) Proteinase K protection assay of mitochondria isolated from HCT116 cells that was transfected with MTS2-FKBP4 and treated with siFKBP4_3′UTR. PK, proteinase K; MM, mitochondrial matrix; IMS, intermembrane space; OMM, outer mitochondrial membrane. TFAM (MM protein), CYCS (IMS protein), and Tom20 (OMM protein) were used as the control showing the extent of digestion. (G, H) Mitochondrial respiration profiles of HCT116 cells transfected with an empty vector (EV) or MTS2-FKBP4 in combination with the treatment of siCtrl or siRNA targeting 3′UTR of endogenous FKBP4 mRNA (siFKBP4). O, oligomycin; F, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP); R+A, rotenone and antimycin A. (H) Basal, ATP-linked respiration, and maximal respiratory capacity were calculated. ***P < 0.001 by the two-tailed t test. Data are presented as mean + SD. n = 6.
Fig 5: ELAM ameliorates mitochondrial function. (A) Representative renal immunofluorescence staining for PINK (green) and Parkin (red). No change in their co-localization showed no mitophagy. (B) Renal oxidative stress was evaluated by in situ production of superoxide anion by fluorescence microscopy using dihydroethidium (DHE), showing increased renal oxidative stress that ELAM ameliorated. (C–E) Mitochondrial function in MetS kidneys. Mitochondrial ATP production (ATP/ADP ratio) decreased in MetS compared to lean kidneys and were restored in MetS + ELAM. H2O2 production increased in MetS kidneys but normalized in MetS + ELAM. Compared to Lean, COX-IV activity was lower in MetS, but not in MetS + ELAM. * p < 0.05 vs. lean, + p < 0.05 vs. MetS, Scale bar = 20 µm.
Supplier Page from Abcam for Complex IV Human Enzyme Activity Microplate Assay Kit