Mitochondrial complex I in-gel activity (IGA) measured in cells expressing wt-CFTR.

<p>A: IGA of mitochondrial extracts from T84 cells after 24 h of treatment with 100 µM glibenclamide or 5 µM CFTR(inh)-172. B: Densitometric quantification of the results shown in panel A, expressed as % ratio of (mCx-I activity)/(protein load). C: IGA of mitochondrial extracts from T84 cells after 48 h of treatment with 100 µM glibenclamide or 5 µM CFTR(inh)-172. D: Densitometric queantification of C. E: IGA of the mCx-I from Caco-2 cells after 48 h of treatment with 5 µM GlyH101 or 5 µM CFTR(inh)-172, and WB of the mCx-III subunit UQCRC1, as internal standard. F: Densitometric quantification of the results shown in E expressed as % ratio of (mCx-I IGA)/UQCRC1(a.u.). The activity of mCx-I in T84 and Caco-2 cells treated with the same amount of DMSO (0.1%) was considered as 100%. Measurements in T84 cells were performed in duplicate and data are expressed as mean ± SE of three independent experiments (n = 3). Caco-2 cells results were obtained in triplicate and expressed as mean ± SE of three independent experiments (n = 3). * indicates p<0.05, as compared with control cells (ANOVA and Turkey's test).</p

Mitochondrial complex I in-gel activity (IGA) of IB3-1 and S9 cells.

<p>A: IGA of mitochondrial extracts from Control and CFTR-stimulated IB3-1 and S9 cells (IBMX-isop-cAMP), adding 200 µM cAMP, 10 µM isoproterenol, 200 µM IBMX, for 24 h. B: Densitometric quantification and statistical analysis of the results shown in panel A. IGA was calculated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048059#pone-0048059-g001" target="_blank">Figure 1</a>. Measurements were performed in duplicate and data are expressed as mean ± SE of five independent experiments (n = 5). * indicates p<0.05, as compared with S9 stimulated cells. C: IGA of mCx-I and mCx-III (UQCRC1) expression measured by using Western blots from S9 and IB3-1 cells (both after CFTR stimulation). D: Densitometric quantification and statistical analysis of the results shown in panel C. IGA of mCx-I was calculated as the ratio mCx-I IGA/UQCRC1. Measurements were performed in duplicate and data are expressed as mean ± SE of two independent experiments (n = 2). * indicates p<0.05, as compared with S9 stimulated cells. E: IGA of the mCx-I and Coomassie blue stain from a BN-PAGE using mitochondrial extracts from S9 and IB3-1 cells. F: Specific activity of the results shown in panel E, calculated as the ratio mCx-I IGA/mCx-I coomassie blue stain. The mCx-I specific activity is expressed in arbitrary units (a.u.) as mean ± SE (n = 3). The specific activity of CF cells (IB3-1) and CF corrected cells (S9) showed similar values, without significant differences (p>0.05).</p

Mitochondrial complex I in-gel activity (IGA) of CFDE and CFDE/6RepCFTR cells.

<p>A: IGA of mitochondrial extracts from CFDE (CF cells), CFDE/6RepCFTR cells (rescued cells ectopically expressing wt-CFTR), and the same cells treated with glibenclamide, a CFTR chloride transport inhibitor. B: Densitometric quantification and statistical analysis of the results shown in panel A. IGA was calculated as the ratio (mCx-I activity)/(protein load), both expressed as arbitrary units. The average activity of the mCx-I in CFDE/6RepCFTR cells was considered 100%. Measurements were performed in duplicate and data are expressed as mean ± SE of three independent experiments (n = 3). ** indicates p<0.01 and *** indicates p<0.001, referred to CFDE/6RepCFTR cells.</p

Stable CFTR knock down and mCx-I activity.

<p>A: CFTR mRNA expression levels in Caco-2/control cells (transfected with pRS control) and Caco-2/pRS26 cells (transfected with the shRNAi pRS26) determined by quantitative real-time RT-PCR. The results were expressed in arbitrary units (a.u.). Measurements were performed in five independent experiments (n = 5) each done in duplicate. B: CFTR channel halide transport activity of pRS control (-•-) and pRS26 cells (-○-). Arrows indicate the points of buffers addition. F, indicates fluorescence values; Fq, are the fluorescence values after SPQ quenching by adding NaI plus valinomicyn buffer (at 750 s). The graph is representative of three independent experiments (n = 3), each done in duplicate. Changes in the halide efflux between pRS control and pRS26 cells, where represented as the areas under the curve (total halide efflux, panel C) and also as the halide efflux slopes (slope of the first 10 points after cAMP stimulation, adjusted by linear regression) (halide efflux rate, panel D). C and D data were plotted as percentage (%) relative to controls. E: Spectrophotometric measurement of the mCx-I activity in CFTR knock down cells compared to control cells, expressed as percentage (%) relative to control values. The cells were incubated 24 h in serum free medium before the experiments. All data were expressed as mean ± SE. ** indicates p<0.001 and *p<0.05, as compared with control cells. Statistical analyses were performed by ANOVA and Turkey's test.</p

Effects of CFTR-shRNAi in T84 cells on the mitochondrial complex I in-gel activity (IGA).

<p>The figure shows the CFTR channel transport activity of T84 cells transiently transfected with a shRNAi plasmid against <i>CFTR</i> and its effects on the IGA of mCx-I. A: T84 cells were transfected with empty plasmids (as control, mock-transfected cells) and shRNAi plasmids, to transiently knock down CFTR. Transfected cells were loaded overnight with 5 mM SPQ (Cl⁻ fluorescent probe) to measure CFTR chloride transport activity. The CFTR activity was measured 24 h (shRNAi: -○-, mock: -•-) and 48 h (shRNAi: -Δ-, mock: -▴-) post electroporation. NaI, indicates perfusion with buffer NaI to quench the SPQ fluorescence at the beginning of the experiment. NaNO₃, indicates the addition of the NaNO₃ buffer to measure the basal activity of the CFTR. cAMP, indicates stimulation of the CFTR activity by adding 200 µM cAMP, 10 µM isoproterenol, 200 µM IBMX in NaNO₃ buffer. NaI plus Valinomicyn, indicates the addition of quenching buffer. F, indicates fluorescence values; Fi, are initial fluorescence values just before adding the NaNO₃ buffer. B: To analyze the CFTR activity changes observed in panel A, the halide efflux was expressed as the area under the curve (integration), expressed as arbitrary units (a.u.). Mock: T84 cells transfected with the empty plasmid as control; shRNAi: T84 cells transfected with the shRNAi plasmid, (24 h and 48 h post transfection). Data are expressed as mean ± SE of two independent experiments (n = 2). * indicates p<0.05 as compared with mock-transfected cells. C: IGA of the mCx-I from mitochondrial extracts corresponding to T84 cells transfected with CFTR-specific shRNAi or empty pSilencer plasmids (Mock). Measurement was performed 48 h post transfection. D: Densitometric quantification and statistical analysis of the results shown in panel C indicated as the ratio (mCx-I activity)/(protein load). Measurements were performed in duplicate and data are expressed as mean ± SE of four independent experiments (n = 4). * indicates p<0.05, as compared with mock-transfected cells (ANOVA and Turkey's test).</p

CFTR modulation and reduced mCx-I activity.

<p>The graphic illustrates the results obtained and possible effects of a reduced activity of mCx-I, according to know relationships extracted from published work by using the Pathway Studio Software (Ariadne Genomics). Small molecules are indicated in green, proteins in red-orange, cellular processes in yellow and diseases in violet. Some relationships found by the program through its curated database were deleted or fused to simplify the illustration and few were added manually using data extracted from PubMed by using the program subroutines (the last relationships shown as solid lines).</p