Figure 2. Depletion of IFTs in vitro impairs ciliogenesis but can also elicit non-ciliary defects in MT networks.

Primary MKs were infected with the lentiviruses indicated, selected for puromycin resistance (viral integration) and then Ca⁺²-shifted 48 hr prior to analyses (A) ShRNA-mediated Ift KDs. Depletion of Ift88 and Ift172, but not Ift74, reduces levels of acetylated tubulin and inhibits Ca⁺²-induced MT reorganization. Arrows point to primary cilia present on MKs expressing scrambled shRNAs but not on cells depleted of IFT74, IFT88 or IFT172. Boxed regions are shown magnified below. Bars, 10µM. (B) Representative immunoblots of lysates from MKs expressing Ift-shRNAs indicated (S, scrambled shRNA). HPRT, loading control. (C, D) Quantifications of ciliogenesis, showing suppression upon IFT-depletion (C) and rescue of ciliogenesis defects upon overexpression of non-targeting Ift74 and Ift88 cDNAs (see methods). Histograms represent data from >2 independent experiments where cilia were quantified in shRNA KD cell lines after Ca⁺²-shift. See also Figure S2.