6533b883fe1ef96bd12dde68

RESEARCH PRODUCT

Additional file 5: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome

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Data supplementary to the nocodazole destabilization assay shown in Fig. 7 . a, b Replicate images of DMSO or nocodazole-treated hTERT-RPE1 cells. Cells were transfected with SF-TAP-tagged KIAA0556 (detected with anti-FLAG immunostaining; green) or GFP-KIAA0556 and counterstained with anti-acetylated tubulin (red) and DAPI (blue). Cells with high KIAA0556 expression are characterised by a filamentous staining pattern and spots of accumulated KIAA0556 signal. In non-transfected cells, 10 minute nocodazole treatment resulted in the loss of a stabilised MT network (see especially the high exposure images), as judged by loss of (almost) all cytoplasmic acetylated tubulin staining and/or the absence of a filamentous staining pattern. In transfected cells (expressing KIAA0556), a filamentous acetylated tubulin staining pattern remained. See also Fig. 7 for examples. Scale bar, 20 μm. c Quantification of the presence of a detectable filamentous acetylated alpha tubulin MT network in GFP-KIAA0556 transfected and non-transfected cells, treated with DMSO or 10 μM nocodazole for 10 minutes. MT networks could be identified in approximately 80 % of GFP-KIAA0556 transfected cells (n = 15) compared with untransfected cells (n > 200). Due to the lower expression level and transfection efficiency of GFP-KIAA0556 (compared with SF-TAP-tagged KIAA0556 in Fig. 7c), only a relatively small number of transfected cells could be analysed. (JPG 1596 kb)