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. 2015 Jun 26;309(5):L475–L487. doi: 10.1152/ajplung.00060.2015

Fig. 6.

Fig. 6.

F508del CFTR-expressing cells display a Cx43-specific trafficking anomaly rescued by 4-phenylbutyric acid (4-PBA). A: NuLi-1 and CuFi-5 cells were fixed and immunostained for Cx43 and Cx26 proteins. Normal appearance of gap junction plaques is shown by punctate staining along cell borders. Cx43 and Cx26 appeared normal in NuLi-1 cells, but Cx43 showed perinuclear localization in CuFi-5 cells. Cx26 localization was unaffected in CuFi-5 cells and appeared normal. Scale bars = 20 μm. B: to assess gap junction function, calcein (0.6 kDa) and Texas Red-labeled dextran (10 kDa) were scrape-loaded into NuLi-1 and CuFi-5 cells grown on filters at week 8 of ALI. CuFi-5 cells were analyzed by fluorescence microscopy after addition of 0, 3, or 5 mM 4-PBA to the basolateral media chamber for 24 h prior to scrape-loading for 15 min at 37°C. In CuFi-5 cells treated with 5 mM 4-PBA, dye transfer was increased, presumably through rescued Cx43 channels. Line plots are representative of the distance data used to calculate values shown in C. C: dye transfer by scrape-loading was quantified using the line scan measure tool in NIH ImageJ to measure distance the dye traveled from the scrape edge on either side of the scrape. Although complete rescue was not achieved, a dose response was observed in cells treated with 5 mM 4-PBA for 24 h, indicating a significant rescue effect on Cx43 gap junction function. #P = 0.001, NuLi-1 vs. CuFi-5 at 0 mM (n = 24, n = 36); **P = 0.012, NuLi-1 vs. CuFi-5 at 3 mM (n = 24, n = 27); *P = 0.032, NuLi-1 vs. CuFi-5 at 5 mM (n = 24, n = 42); *P = 0.050, CuFi-5 at 0 mM vs. CuFi-5 at 5 mM (by 2-tailed Student's t-test). D: effect of 4-PBA rescue on F508del CFTR Cl− currents. Ussing chamber experiments were conducted in 8-wk-old CuFi-5 cell filters basolaterally treated with 0, 3, or 5 mM 4-PBA for 24 h before analysis for forskolin and IBMX (F + I)-induced currents. Changes in short-circuit currents (ΔIsc) for induction and inhibition experiments indicate that 4-PBA had a positive effect on CFTR-mediated Cl− currents (n = 4 filters each). *P = 0.022, ^P = 0.064, ^^P = 0.055 (by 2-tailed Student's t-test).