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. 2018 Sep 3;217(9):3255–3266. doi: 10.1083/jcb.201703196

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© 2018 Drummond et al.

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Figure 3. — Cdc42 regulates primary cilia and Hh signaling by recruiting aPKC–Par6a to the basal body. (A) Immunofluorescence of mDCs stained for Toca1 or Cdc42 with acetylated-tubulin (AcTub) – or adenyl cyclase III (ACIII)–positive primary cilia. Immunoreactivity around the basal body is highlighted in the lower right of each panel. Bars: 10 µm; (inset) 2 µm. (B and C) Percentage of subconfluent mDCs displaying primary cilia (B) and violin plot of axoneme lengths (C) after shRNA knockdown of control (n = 4 experiments), Toca1 (n = 3 experiments), or Cdc42 (n = 4 experiments). sh, short hairpin. (D) Gli1 mRNA levels of confluent mDCs after shRNA knockdown of control (n = 9 experiments), Toca1 (n = 2 experiments), or Cdc42 (n = 3 experiments) and treated with control-conditioned media or Shh-CM. CM, conditioned media. dR, delta reporter signal normalized to passive reference dye. (E and F) Percentage of subconfluent mDCs displaying primary cilia (E) and violin plot of axoneme lengths (F) after shRNA knockdown of control (n = 4 experiments), aPKC (n = 4 experiments), Par6a (n = 4 experiments), or Par3 (n = 4 experiments). sh, short hairpin. (G) Gli1 mRNA levels of confluent mDCs after shRNA knockdown of control (n = 9 experiments) or aPKC (n = 6 experiments) and treated with control-conditioned media or Shh-CM. KD, knockdown. Error bars represent SEM. Significance determined by unpaired two-tailed t test (**, P < 0.01; ***, P < 0.001). (H) Immunofluorescence of control, aPKC, Par6a, Cdc42, or Par3 knockdown mDCs stained for indicated proteins and AcTub/ACIII–positive primary cilia or pericentrin/γ-tubulin (gtub)–positive basal bodies. Immunoreactivity around the basal body is highlighted in the lower right of each panel. Bars: 10 µm; (inset) 2 µm. (I) Western blot of control, Cdc42, or Par3 knockdown mDCs that are probed for polarity proteins and actin.