When we analysed the expression and localisation of the Cx43 protein by epifluorescence and confocal microscopy in the three cell types grown on Transwells for 6 days, the XY images showed a similar signal in CFBE and 16HBE cells, localised at the cell borders (Figure 1(b)).
Since the target of downregulation studies is the coculture of hAMSCs and CFBE cells, cocultures at 1: 5 hAMSC: CFBE ratio was used in this assay.
We confirmed confocal microscopy results by Western blotting analysis performed in 16HBE (endogenously expressing wt CFTR and therefore used as a reference to the location of band C and band B of wt CFTR), CFBE, hAMSCs, and cocultures transfected with either siRNA for Cx43 or scrambled siRNA grown on Transwells.
Importantly, the active siRNA transfection resulted in a significant reduction of chloride efflux in comparison to the other two conditions, suggesting a central role of GJs in the cross-talk between hAMSCs and CFBE cells and the rescue of CFTR protein expression and function.
In CFBE cells, CFTR overexpression has been shown to increase both the CFTR trafficking to the plasma membrane and the transepithelial resistance (TER) while reducing the permeability to small molecules .
Although there was a significant decrease in cell viability in control cocultures and cocultures transfected with scrambled siRNA as compared with CFBE single cultures (Supplementary Table 1), the percentages of viable (7-AAD(-) and annexin V(-)), necrotic (7-AAD(+) and annexin V(+)), early apoptotic (7-AAD(-) and annexin V(+)), and late apoptotic (7-AAD(+) and annexin V(-)) cells were not different among control cocultures and cocultures transfected with either Cx43 or the scrambled siRNA.
This study underlines the relevance of GJs in the rescue of CFTR protein expression and function as a chloride channel in CFBE: hAMSCs cocultures.
Our previously published data  strongly suggested that intercellular communication is involved in the rescue of CF-associated basic defects by hAMSCs in coculture with CFBE cells.
These results are in agreement with our previous results showing that hAMSC: CFBE cocultures displayed a corrected CFTR protein in association with an increase in ZO-1 localisation at the intercellular borders .
Cocultures of hASMCs and CFBE (1:5 ratio) were transfected with a FITC-conjugated siRNA (see Materials and Methods).
(a) mRNA levels: CFBE, 16HBE, and hAMSCs were grown on Transwells for 6 days at confluence and then evaluated by real-time PCR.
1:5 hAMSC: CFBE cocultures were transfected with either Cx43 siRNA or scrambled siRNA for 6 days.