Figure 4. Effects of CFTR-shRNAi in T84 cells on the mitochondrial complex I in-gel activity (IGA).

The figure shows the CFTR channel transport activity of T84 cells transiently transfected with a shRNAi plasmid against CFTR 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).