Generation of MEFs
E12.5 mouse embryos were dissected, and the head and visceral organs were removed. The embryos were then washed in clean phosphate-buffered saline (PBS), and single embryos were each transferred to a 1-ml syringe. The embryos were passed through an 18-G needle multiple times in DMEM (high glucose, 10% fetal bovine serum [FBS], penicillin, streptomycin) and transferred to a gelatinized 10-cm tissue culture dish. After the cells reached confluency, they were split 1:5. Cells were not used after passage 4.
Cells were grown on coverslips and fixed 10 min in 4% paraformaldehyde (PFA) in PBS, then washed in antibody wash buffer (PBS, 0.1% TritonX-100, 1% heat-inactivated goat serum) for 10 min at room temperature. Primary antibodies were incubated overnight at 4°C, and after a series of washes, they were incubated in secondary antibody for 1 h at room temperature. After a second series of washes, coverslips were mounted on slides with ProLong Gold antifade reagent (Invitrogen, Carlsbad, CA). Antibodies and dilutions were as follows: Ptch1 (1:250; Raj Rahatgi, Stanford University, Stanford, CA), Smo (1:500; Kathryn Anderson, Sloan-Kettering Institute, New York, NY), acetylated α-tubulin (1:2500; T-6793, Sigma-Aldrich, St. Louis, MO), γ-tubulin (1:1000; T-6557, Sigma), Gli2 (1:2000; Jonathan Eggenschwiler, Princeton University, Princeton, NJ), Gli3C and Gli3N (2 μg/ml; Suzie Scales, Genentech, San Francisco, CA; Wen et al., 2010
), polyclonal GFP (1:500, AB3080, Millipore, Billerica, MA), monoclonal GFP (1:500, MAB3580, Chemicon), Sufu (1:100, SC-10933, Santa Cruz Biotechnology, Santa Cruz, CA), glutamylated tubulin (GT335, Carsten Janke, Curie Institute, Paris), and Arl13b (1:1500).
Fluorescence intensities were measured using ImageJ software, and were normalized to cell-body staining. All cilia in a field were measured, and cilia were selected in the acetylated α-tubulin channel to prevent biased selection. For all antibodies except Gli2, Gli3 C-terminal, Gli3 N-terminal, and Sufu, the average intensity in the entire cilium was measured relative to background. For the Glis and Sufu, which only stain the tips of cilia, the fluorescence intensity in only the tip was measured relative to background. The tip of the cilium was determined by weakened acetylated α-tubulin staining, as well as orientation. Three independent experiments were performed for all antibodies, and a total of at least 125 cilia were measured for the Gli and Sufu antibodies, and at least 40 cilia were measured for all other antibodies. Significance was determined for all experiments using a one-tailed Student's t test. Smo and Ptch1 antibodies were imaged on a Zeiss LSM510 META (Carl Zeiss Microscopy, Thornwood, NY) confocal at 63× with optical zoom. All other antibodies were imaged on a Leica DM6000B (Leica Microsystems, Buffalo Grove, IL) upright microscope at 100× with SimplePCI software (Hamamatsu Corp., Sewickley, PA).
For removal of the ciliary membrane, cells were treated with 0.1% TritonX-100 in PBS without Ca2+ or Mg2+ for 1 min and then fixed in 4% PFA. Antibody staining was then performed as described in the preceding paragraphs.
IMCD3 cells were transduced with a viral construct expressing Arl13b-GFP. All cells were grown to confluency. SSTR3-GFP cells were from Greg Pazour and IFT88-YFP cells were from Brad Yoder. FRAP was performed on a Zeiss LSM510 META confocal using 63× objective with optical zoom. Briefly, cilia were photobleached using the 488-nm laser with 25 iterations at 50% power. Images were scanned at 3% laser power. Fluorescence intensity measurements had background subtracted, and the bleached region was normalized to the entire cilium (for determination of movement within the cilium) or to an unbleached region in the same field (for determination of turnover in the cilium).
Conditioned medium was generated as previously described (Taipale et al., 2000
). Briefly, 293 EcR Shh cells (ATCC; CRL-2782) were grown to confluence, and 1 μM of MurA was added to the cells in 2% serum DMEM. After 24 h, the conditioned medium was collected and the 2% serum MurA media was replaced. A collection was taken again after 24 h, and that medium was combined with the first collection.
For immunofluorescence of MEFs treated with Shh-conditioned media, cells were plated at 800,000 cells per well of a six-well plate. At confluence, cells were serum-starved in DMEM high glucose with 0% serum. After 24 h of serum-starving, conditioned medium diluted 1:4 was added to the cells. Untreated control cells were given 0.5% serum media at this point. After 24 h of treatment, the cells were harvested for immunofluorescence.
The knockdown viral construct was generated using Sigma's Mission custom viral vector synthesis. The vector, clone ID, and targeting sequence were pLKO.1-puro-CMV-TagRFP, TRCN0000100504, and CCTGTCAGAAAGGTGACACTT, respectively. The targeting sequence targets the coding region of Arl13b starting at nucleotide 349 of the mRNA.
IMCD3 cells were transduced in a 12-well plate with the knockdown construct at a multiplicity of infection of 5. After transduction, cells were treated with puromycin at 2 μg/ml for 3 d. After treatment, cells were passaged to a six-well plate for immunofluorescence or to be harvested for Western blot analysis.
Arl13b constructs and overexpression
Arl13b was cloned using the Gateway system (Invitrogen). Full-length Arl13b (coding for amino acids 1–427 without stop codon), the N-terminal domain (coding for amino acids 1–212 of Arl13b), and the C-terminal domain (coding for amino acids 210–427 of Arl13b) were cloned into pENTR/SD/D-TOPO (K242020, Invitrogen). The inserts were recombined into the pcDNA-DEST47 (C-terminal GFP tag; 12281010, Invitrogen) and pcDNA-DEST40 (N-terminal GFP tag; 12274015, Invitrogen) using Gateway LR Clonase (11791-019, Invitrogen) following the manufacturer's instructions.
Cells (350,000 per well) were seeded in a pregelatinized six-well plate containing coverslips in DMEM (high glucose, 10% FBS, penicillin, streptomycin). After 24 h, the cells were transfected following the manufacturer's recommendations using Lipofectamine 2000 (Invitrogen) complexed with the Arl13b expression plasmids. After a 5-h incubation period of cells with the complexes, the medium was changed to serum-free DMEM High Glucose for 24 h. The cells were then fixed using 4% PFA and were subjected to immunofluorescence. Cilia were measured using LSM Image Examiner software (Carl Zeiss Microscopy, Thornwood, NY).
Arl13b-GFP expression virus
The L13 lentiviral mammalian expression vector was obtained from the Emory University Viral Vector Core. The Arl13b-GFP insert was generated by PCR from the pcDNA-DEST47 plasmid containing full-length Arl13b and using the following primers (5′ to 3′): forward- GCTAGCTCAAACAAGTTTGTACAAAAAAGC, reverse- GGATCCATTCTAGATCGAACCACTTTG. The primers introduced a 5′ NheI site and a 3′ BamHI site that were used to insert Arl13b-GFP into L13.