Fig. 4. 18 GA inhibits GJIC and downregulates ß-casein expression by CID-9 cells on day 5 of culture in differentiation medium. (A) Cells were plated on EHS-drip in the absence (Ctrl; control) or presence of 10 µM 18 GA (18G). LY dye transfer in scrape-loaded cells was markedly affected by 18 GA. Integrated fluorescence intensity, quantified from three different experiments as described in Materials and Methods, showed a threefold decrease. Western blot analysis showed that 18 GA downregulated Cx43 and ß-casein levels without drastically affecting cell viability as determined by Trypan Blue staining. (B) Morphology of CID-9 cells plated in differentiation medium on EHS-matrix on day 6 of culture (a), compared with cells treated with 10 µM 18 GA (b). Note the partial restoration of normal clustering morphology 4 days (i.e. day 10 of culture) after 18 GA removal (c). Western blot analysis, normalized to equal cell count, showed that ß-casein was partially recovered (approximately 50% recovery) upon 18 GA withdrawal.