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. 2021 Apr 15;220(5):e202007207. doi: 10.1083/jcb.202007207

Figure 1.

Figure 1.

Modest Cilia-APEX2 expression enables labeling of the ciliary contents without disturbing the ciliary localization of Hh signaling components. (A) Diagram of Hh signaling. Positive and negative regulators are in green and red boxes, respectively. Pharmacological agents are in oval circles and proteins in rectangles. Acronyms are defined in the text. (B) Diagrams of the Cilia-APEX and Cilia-APEX2 expression cassettes. Numbers indicate amino acid positions. The N terminus of NPHP3 is myristoylated at glycine 2 (Myr) and confers ciliary targeting. The truncated CMV promoter (pCMV(Δ6)) is considerably weaker than the EF1α promoter (pEF1α). (C) IMCD3 cells and stable clones expressing Cilia-APEX or Cilia-APEX2 were serum-starved for 24 h in the presence or absence of Shh before fixation and staining for GPR161 (white) and ARL13B (red). Cilia-APEX and Cilia-APEX2 were visualized via the intrinsic fluorescence of GFP (green). DNA in blue. (D) Box plots showing the relative GPR161 fluorescence normalized to ARL13B in the primary cilium of IMCD3, Cilia-APEX, and Cilia-APEX2 cell lines after Shh treatment as in A. n = 50 cilia per condition. In these and all subsequent box plots, crosses indicate mean values, whiskers indicate values within 1.5× interquartile range, and dots represent outliers. RFU, relative fluorescence unit. (E) IMCD3, Cilia-APEX, and Cilia-APEX2 cell lines were immunoblotted for GFP (APEX fusions), IFT88, and actin. (F) Signals from immunoblots as in E were quantified, ratios of GFP signals in the Cilia-APEX relative to the Cilia-APEX2 cell line were calculated, and results plotted. Thick horizontal line represents the mean (n = 3). (G) IMCD3, Cilia-APEX, and Cilia-APEX2 cell lines were subjected to APEX labeling before fixation and staining for ARL13B (red) and biotin (white). The APEX fusion proteins are detected via intrinsic GFP fluorescence (green). DNA in blue. (H and I) Box plots showing background-subtracted intensities of GFP (H) and biotin (I) signals in the primary cilium from images as in G. n = 30 cilia per condition. Scale bars, 2 µm in all panels.