Figure 6
AFTPH overexpression increased NTR1 levels in human colonic epithelial cells treated by Bafilomycin A1.
(A) MiR-133α gene silencing and AFTPH overexpression in NCM460-NTR1 cells did not affect NTR1 levels in human colonic epithelial cells when compared with their respective controls under the same treatment (unstimulated or upon 3 hr after recovery), as examined by In-cell ELISA. (B) Treatment with Bafilomycin A1 (BafA1, 100 nM), a specific vacuolar H+-ATPase inhibitor, increased NTR1 levels in as-miR-133α-transfected or AFTPH-overexpressing cells when compared to their respective controls. Similar effects were not observed in cells treated with Brefeldin A (BFA, 5 μg/mL). **p < 0.01 when compared with cells without incubating with inhibitors during recovery. (C) Bafilomycin A1 and Brefeldin A treatment during recovery blocked NTR1 recycling in both NCM460-NTR1 cells and their AFTPH-overexpressing counterparts when compared to their respective controls, as visualized in immunocytochemistry. Scale bars, 10 μm. (D) Intracellular NTR1 expression was increased in NCM460-NTR1-AFTPH cells treated with Bafilomycin A1, when compared to NCM460-NTR1 cells under the same treatment as examined in quantitative image analysis. Incubation with Brefeldin A during recovery did not generate similar effect on the two cell lines. *p < 0.05 when compared with cells treated with vehicle controls.
