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. 2021 Jan 1;32(1):45–56. doi: 10.1091/mbc.E20-08-0556

FIGURE 4:

FIGURE 4:

Defects in ciliary localization of the IFT components and ARL13B in IFT88(Δα)-expressing IFT88-KO cells. (A–E) IFT88-KO cells stably expressing mChe as a negative control (A) or mChe-fused IFT88(WT) (B), IFT88(ΔNT) (C), IFT88(ΔCT) (D), or IFT88(Δα) (E) were serum-starved for 24 h to induce ciliogenesis, and immunostained for mChe (A–E), ARL13B (A′–E′), and Ac-tubulin+FOP (A′′–E′′). Scale bar, 5 µm. (F) Ciliated IFT88-KO cells expressing mChe or the mChe-fused IFT88 constructs shown in A–E were counted, and percentages of ciliated cells are expressed as bar graphs. Values are means ± SD of three independent experiments. In each set of experiments, 33 to 56 cells were analyzed, and the total numbers of cells analyzed (n) are shown. The p value was determined by the Student t test. (G, M) The length of cilia (G) and ciliary ARL13B staining intensity (M) in the IFT88-KO cells expressing mChe-IFT88(WT) or mChe-IFT88(Δα) were measured, and the ciliary length and the ciliary ARL13B staining intensity per length are represented as box-and-whisker plots. The boxes represent the 25th–75th percentiles (interquartile range [IQR]), and the median is indicated by a horizontal line. The whiskers show the minimum and maximum within 1.5 ×  IQR from the 25th and 75th percentiles, respectively. Outliers are indicated as dots. The total numbers of ciliated cells analyzed (n) are shown. P values were determined by the Student t test. (H–K) IFT88-KO cells stably expressing mChe-IFT88(WT) (H, J) or mChe-IFT88(Δα) (I, K) were immunostained for either IFT57 (H′, I′) or IFT140 (J′, K′), mChe (H–K), and Ac-tubulin and FOP (H′′–K′′). Scale bar, 5 µm. (L) Localization of IFT140 was classified as “ciliary base” or “ciliary base and tip,” and the cells in each category were counted. The percentages of these populations are represented as stacked bar graphs. Values are the means of three independent experiments. In each set of experiments, 30–37 ciliated cells were observed, and the total numbers of ciliated cells observed (n) are shown. The p value was determined by the Pearson χ2 test.