JCS037408 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Establishments of wild-type, inv and Inv-GFP cell lines. (A) Identification of proximal convoluted tubule cells and collecting duct cells by lectin staining. Binding of lectins to mouse kidney sections, wild-type and inv mice are shown. LTA-FITC conjugate gives a strong reaction with proximal convoluted tubule cells (green) and DBA-rhodamine conjugates with collecting duct cells (red). Scale bar: 200 µm. (B) Isolation of proximal convoluted tubule cells using LTA-FITC as a marker. Fluorescent signals from FITC-positive cells are ten times higher than from FITC-negative cells. Proximal convoluted tubule cells were sorted by flow cytometry (FCM). (C) Confirmation of genotypes. Genotypes were determined by PCR analysis. Primer pairs for Inv used to detect wild-type locus, tyrosinase to detect Inv locus, and GFP to detect the Inv-GFP transgene. These cell lines are named wild-type cells (clone Dai1), inv cells (clone Dai2), and Inv-GFP cells (clone Dai3), respectively. (D) Live-cell images of established cell lines. Primary cilia in living cells are seen as a dot. Scale bar: 10 µm. (E) Primary cilia staining of established cell lines. Primary cilia were detected by anti-acetylated α-tubulin antibody (red). All established cell lines process long primary cilia. Scale bar: 20 µm. (F) Signal intensities of endogenous Inv and Inv-GFP protein in the primary cilia. Wild-type cells and Inv-GFP cells were co-cultured until primary cilia were formed. Inv-GFP cells do not express endogenous Inv. Both Inv-GFP protein in Inv-GFP cells and endogenous Inv in wild-type cells were detected by anti-Inv antibody and Alexa-Fluor-555-conjugated goat anti-rabbit IgG (red). Wild-type cells (*) and Inv-GFP cells (#) were easily distinguished by Inv-GFP signal (green). Scale bar: 10 µm. Fluorescent intensity (Alexa Fluor 555) of Inv-GFP in Inv-GFP cells was almost identical to fluorescent intensity (Alexa Fluor 555) of endogenous Inv in wild-type cells. Signal intensity of Inv-GFP protein was expressed as fold-increase compared with endogenous Inv signal (means ± s.e., n=10), and was shown on the right.

• Supplemental Figure S2 -

  Fig. S2. Centrosome localization of Inv (1001-1062) fusion proteins during the cell cycle. GFP-tagged Inv (1001-1062) signal (green) was detected at the centrosome, confirmed by anti-γ-tubulin antibody (red) in interphase. Localization of the fusion protein to spindle pole was evident through prophase to metaphase. In anaphase, weak signal at spindle pole was detected, but by telophase, the signal had completely disappeared. Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. Inv was extracted from the axoneme under NP-40 treatment. Inv-GFP cells were incubated with culture medium containing propidium iodide (PI, a fluorescent nucleic acid dye; 1 µg/ml) in the absence (A) or presence (B) of NP-40 (0.25%) for 90 seconds at room temperature, and then fixed. PI staining (red) is used for evaluating the disruption of cell membrane since PI is membrane impermeable. Primary cilia were detected by anti-acetylated α-tubulin antibody (blue). GFP-tagged Inv protein signals (green) disappeared under the NP-40 treatment. Scale bar: 10 µm.

• Movie 1 - Movie 1. Live image of primary cilium and Inv-GFP fluorescence. Primary cilium and Inv-GFP fluorescence are observed while moving the focus. The focal level from the cell surface is indicated under the left from 0 to 9.5 µm. Successive pictures are combined and formatted as a movie file. The primary cilium is seen as a dot in living cells viewed from the top, thus it is difficult to distinguish the cilium from cell debris. However, it was easily and clearly identified by moving the focus live. Inv-GFP fluorescence was predominantly detected at the base of the primary cilium.