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. 2015 Dec;53(6):853–862. doi: 10.1165/rcmb.2014-0462OC

Figure 6.

Figure 6.

Decreased tubulin polymerization–promoting protein (TPPP) 1 expression leads to CF phenotypes. Expression of TPPP1 was successfully knocked down in non-CF human tracheal epithelial (9/HTEo−) cells, which were stably transfected with short hairpin RNA (shRNA) targeting TPPP1 (shTPPP1). (A) Lysates from control 9/HTEo− cells with scrambled sequence (9/HTEo− Neg Ctrl) and 9/HTEo− with TPPP1 shRNA (9/HTEo− shTPPP1) were analyzed via Western blot for acetylated tubulin (Ac-tub), TPPP1, and Ras homolog gene family, member A (RhoA). Representative blots are shown along with quantification to the right (TPPP1/actin, RhoA/actin, and Ac-tub/α-tub). Significance was determined by t test (RhoA/actin, n = 11, *P < 0.01; TPPP1/actin, n = 11, *P < 0.01; Ac-tub/α-tub, n = 11, *P < 0.01). (B) 9/HTEo− Neg Ctrl and 9/HTEo− shTPPP1 cells were subjected to a microtubule polymerization assay and immunostained for α-tubulin. Quantification of microtubule polymerization assay for 9/HTEo− Neg Ctrl and 9/HTEo− shTPPP1 cells was determined by the ratio of cells with asters to total cells at various time points. Significance was determined by t-test comparing 9/HTEo− Neg Ctrl to 9/HTEo− shTPPP1 ratios at various time points (0–20 min; n = 4; *P < 0.05). (C) 9/HTEo− Neg Ctrl and 9/HTEo− shTPPP1 cells were immunostained with Ac-tub antibodies (left). Cells were stained with filipin and quantified by determining percentage of cells with perinuclear cholesterol accumulation (*P < 0.01; right). Representative images are shown from 40 separate images for each cell model. Data represent the mean (±SEM).