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Fig. 5
Effect of INPP5B CAAX mutant on cilia localization.
(A) Domain structure of human INPP5B protein. (B) hTERT-RPE1 cells were transduced by GFP-Inpp5b or GFP-Inpp5bΔCAAX lentivirus, starved for 48 hr, and then analyzed by immunostaining with anti-acetylated alpha-tubulin antibody. Scale bar 10 micron. (C) Lengths of primary cilia in Inpp5b and Inpp5b-delta-CAAX cells. hTERT-RPE1 cells were transduced with either GFP-Inpp5b or GFP-Inpp5b-delta-CAAX lentivirus, serum starved for 48 hr, and stained with anti-acetylated alpha-tubulin antibody. Scatter plot showing distribution pattern of ciliary length (3.4±0.4 micron in INPP5B and 2.8±0.3 micron in Inpp5bΔCAAX cells, unpaired t-test, p = 1.36E-06, n >160 cilia, three independent experiments). (D) INPP5BΔCAAX mRNA failed to rescue the loss of inpp5b. KV cilia of zebrafish embryos injected with inpp5b MO (4 ng), inpp5b MO (4 ng) with INPP5B WT mRNA (500 ng) and inpp5b MO (4 ng) with INPP5B ΔCAAX mRNA (500 ng) at 6-somite stage were immunostained with acetylated α-tubulin (red), representative images are shown (dash line indicates border of KV). Scale bar 10 micron. (E–F) Quantification of length (E) and number (F) of KV cilia in zebrafish embryos injected with inpp5b MO (4 ng), inpp5b MO (4 ng) with INPP5B WT mRNA (500 ng) and inpp5b MO (4 ng) with INPP5B ΔCAAX mRNA (500 ng). (N >20 embryos, three independent experiments, unpaired t-test, * p = 3.9E-26 in E and * p = 9E-06 in F).