Data on cytochrome c oxidase assembly in mice and human fibroblasts or tissues induced by SURF1 defect

This paper describes data related to a research article entitled “Tissue- and species-specific differences in cytochrome c oxidase assembly induced by SURF1 defects” [1]. This paper includes data of the quantitative analysis of individual forms of respiratory chain complexes I, III and IV present in SURF1 knockout (SURF1−/−) and control (SURF1+/+) mouse fibroblasts and tissues and in fibroblasts of human control and patients with SURF1 gene mutation. Also it includes data demonstrating response of complex IV, cytochrome c oxidase (COX), to reversible inhibition of mitochondrial translation in SURF1−/− mouse and SURF1 patient fibroblast cell lines.


Specifications
Tissue-and species-specificity of COX biogenesis at normal and pathological conditions. Reversible mitochondrial translation arrest for analysis of newly synthesized COX in mouse/human fibroblasts.
Approach to study different assembly defects of respiratory chain complexes containing mtDNAencoded subunits.

Data
In the present work related to [1], we show differences in amounts of individual forms of respiratory chain complexes I, III and IV quantified from western blots of 2D BNE/SDS PAGE analysis, as determined in mitochondria of SURF1 þ / þ and SURF1 À / À mouse fibroblasts and tissues (heart, muscle, brain, liver) and also in mitochondria of human control and SURF1 deficient fibroblasts (Figs. 1-3).
Then we show data ( Fig. 4) from analysis of fibroblast cell lines from SURF1 À / À mouse, SURF1 patient and controls, in which translation of mitochondrial DNA encoded proteins was reversibly inhibited with doxycycline (DOX). After DOX removal, the formation of newly synthetized COX in time (0-96 h) was assessed by SDS PAGE and western blot analysis.

Experimental material
For experiments different tissues were obtained from 3-month old SURF1 À /À knockout B6D2F1 mice [2], generated by the insertion of a loxP sequence in exon 7 of the mouse SURF1 gene, leading to an aberrant, prematurely truncated and highly unstable protein, and from control wild type SURF1 þ / þ mice. Immortalized skin fibroblasts from control and SURF1 À /À mouse were cultured at 37°C in 5% atmosphere of CO 2 in a DMEM medium supplemented with 10% fetal bovine serum, 20 mM HEPES (pH 7.5) and geneticin (50 mg/ml). The same conditions were used for cultivation of human patients' skin fibroblasts lacking the SURF1 protein due to 845 del CT mutations of SURF1 gene [3] and from controls, except that geneticin was replaced with penicillin (10 mg/ml) and streptomycin (10 mg/ml). The project was approved by the ethics committees of Institute of Physiology, CAS. Informed consent was obtained from the parents of the patients according to the Declaration of Helsinki of the World Medical Association.
Liver mitochondria were isolated from 10% homogenate prepared in STE medium (250 mM sucrose, 10 mM Tris, 2 mM EDTA, pH 7.2) supplemented with PIC (1:500) using glass-teflon homogenizer (600 rpm, 7 strokes). The homogenate was then centrifuged for 10 min at 800g. Postnuclear supernatant filtered through a gauze was centrifuged for 15 min at 5200g, pelleted mitochondria were washed twice (13,000g, 10 min) in STE with PIC and then resuspended in STE medium.
Heart mitochondria were isolated essentially as liver mitochondria, except that postnuclear supernatant was centrifuged for 10 min at 13,000g.
All isolations were performed at 4°C, mitochondria and cell membranes were stored at À 80°C. Protein concentration was measured according to [6].
Samples for SDS PAGE from 10% (w/v) cell suspension and solubilizates from cell membranes were prepared by adding the same volume of SLB2x lysis buffer and loaded on a 10% slab gel [8].

Western blot analysis
Proteins were transferred from the gels to PVDF membranes (Immobilon-P, Millipore) using semidry electroblotting. The membranes were blocked with 5% (w/v) non-fat dried milk in TBS (150 mM NaCl, 10 mM Tris, pH 7.5) for 1 h and incubated 2 h or overnight at 4°C with primary antibodies diluted in TBS with 0.1% Tween-20. Monoclonal primary antibodies to the following enzymes of OXPHOS were used: SDHA (ab14715, Abcam), CORE1 (ab110252, Abcam), NDUFB6 (ab110244, Abcam), NDUFS3 (ab110246, Abcam), COX1 (ab14705, Abcam). The detection of the signals was performed with the secondary Alexa Fluor 680-labeled antibody (Life Technologies) using the Odyssey fluorescence scanner (LI-COR). Quantification of detected signals from SDS PAGE and BNE/ SDS PAGE was carried out in Aida Image Analyzer program, version 3.21. Fig. 4. Decreased COX1 amount after DOX treatment. Homogenates from DOX treated human control and SURF1 patient fibroblasts (A, B), SURF1 þ / þ and SURF1 À / À mouse fibroblasts (C, D) and digitonin solubilizates from DOX treated human control and SURF1 patient fibroblasts (E, F), SURF1 þ / þ and SURF1 À / À mouse fibroblasts (G, H) were analyzed on SDS PAGE in combination with western blot to obtain overall COX1 signal at different time points (0-96 h) after DOX treatment. Signal of SDHA was used as reference. Control cells without DOX treatment (C -DOX ).

Doxycycline treatment of the cells
Experiment was performed as described in [9]. Briefly, fibroblasts (grown to 70% confluence in DMEM medium) were treated with 15 mg/ml doxycycline (DOX) for 7 days and then washed 3 times with PBS to withdraw DOX. Subsequently, the cells were collected at different time points (0, 6, 16, 24, 48, 72 and 96 h) after DOX removal. Weighed pellets of cells were stored at À 80°C. Two independent experiments of DOX inhibition in human and mouse fibroblasts were performed. The total COX1 signal for each given time point was quantified from 1D SDS PAGE using Aida Image Analyzer version 3.21 (Raytest) and normalized to SDHA signal.