Cell Lines and Cell Culture
(RRF427) E14 ES cell line was obtained from BayGenomics. Ofd1rev
, and cell lines with human mutations were created as described previously (Singla et al., 2009
). Cells were cultured on 0.1% gelatin in GMEM supplemented with 10% FBS, glutamine, pyruvate, NEAA, βME, and LIF.
3T3 (ATCC) and POC1-GFP U2OS (gift of Dr. Wallace Marshall) cells were cultured in DMEM supplemented with 10% FBS and antibiotics. IMCD3 (ATCC) and hTERT-RPE1 (gift of Dr. Wallace Marshall) were cultured in DMEM:F12 supplemented with 10% FBS and antibiotics.
cDNA constructs and cloning
cDNA was cloned as described previously (Singla et al., 2009
). Missense mutations were created using Quik Change II XL site directed mutagenesis kit (Stratagene). Final products were confirmed by sequencing.
Creation of Floxin cell lines
Missense mutations S75F and A80T occur in exon 1 of Ofd1, while G139S occurs in exon 3. The gene trap insertion in Ofd1Gt cells is in intron 3 of the Ofd1 genomic locus. Full length cDNA for Ofd1-Myc-S75F, Ofd1-Myc-A80T, and Ofd1-Myc-G139S, alleles in which the mutation occurs in exons upstream of the gene trap insertion site, was cloned into the vector pFloxin-IRES (Genbank EU916835). Missense mutations S437R and KDD359-361FSY occur in exons downstream of the gene trap insertion site. cDNA for exons 4-23 of Ofd1-Myc-S437R and Ofd1-Myc-KDD359-361FSY was cloned into the vector pFloxin (Genbank EU916834). pFloxin and pFloxin-IRES constructs were electroporated into Ofd1Rev cells as previously described. Cells were selected with 300 μg/mL G418 (Invitrogen) and colonies were transferred to 48 well plates after 6 days. Correct integration was verified by genomic PCR.
Antibodies to Ofd1 were generated by Covance, Inc. Rabbits were immunized with the peptide [H]-CDTYDQKLKTELLKYQLELKDDYI–[NH2] corresponding to amino acids 340-362 of murine Ofd1. Antibody was used at 1:5000 for Western blotting and for immunofluorescence, 1:2000 in murine cells, 1:1000 in human cells. See supplemental experimental
procedures for other antibody information.
Immunofluorescence and Microscopy
For ES cell ciliation studies: ES cells were plated on coverslips coated with 1% matrigel (BD) and treated with 0.5 mM mimosine (Sigma) overnight to arrest cells. Cells were fixed 5′ in 4% PFA, washed in PBS, and fixed 2–3′ in −20° 100% methanol. The cells were washed in PBS with 0.1% Triton-X100 (PBST), blocked in 2% BSA in PBST, and incubated with primary antibodies in block for 1 hr at RT. The cells were washed in PBST, incubated with secondary antibodies in block for 30′ at RT, and mounted with Vectashield hardset with DAPI (Vector labs).
POC1-GFP U2OS S-phase arrest: Cells were plated on coverslips and treated with 3.2 μg/mL aphidocolin (Sigma) for 72 hr, then fixed in 100% methanol and washed and processed as above.
For cell synchronization studies: Cells were synchronized using thymidine-mimosine block(Fujii-Yamamoto et al., 2005
). Briefly, cells were plated on coated coverslips in 2.5 mM thymidine, incubated for 12 hr, released into regular media for 6 hr, then blocked in 0.5 mM mimosine for 6 hr. Timepoints were taken after release from mimosine block. Cells for FACS were collected as described below. For IF, cells were fixed in 100% methanol, then washed and processed as above.
For all other experiments, cells were plated on coverslips and fixed in 100% methanol, then washed and processed as above.
Slides were viewed on a Deltavision microscope (Applied Precision) and image processing was completed with Deltavision and Metamorph (Molecular Devices) software. Images are maximum projections of Z-stacks.
Immunoblots and Quantification
Cells were grown in flasks, trypsinized, collected, and washed once in PBS. Cell pellets were lysed in buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1:200 dilution protease inhibitor cocktail (Calbiochem)) containing 1% NP-40, 1%Triton-X-100, 0.25% sodium deoxycholate, or 1%NP-40 and 0.25% sodium deoxycholate (modified RIPA) for 30 minutes at 4 degrees. Lysates were centrifuged for 15 minutes, 16,000 rcf, at 4 degrees. Cleared supernatants were transferred to a new tube and 6X reducing sample buffer was added to the pellet.
Ofd1 protein expression was quantified by densitometry and normalized to actin.
Cells were grown in flasks, trypsinized, collected, and washed once in PBS. Cell pellets were lysed in modified RIPA buffer for 30 minutes at 4 degrees. Lysates were centrifuged for 15 minutes, 16,000 rcf, at 4 degrees. Protein concentration of the cleared supernatant was determined by Bradford assay. Supernatants were standardized to 1.6 mg/mL concentration, 3 mg total protein, and pre-cleared with protein G agarose beads (Invitrogen) for 2 hours. Beads were removed and supernatants were incubated overnight with 1.6 μg Ofd1 antibody. Next day, complexes were captured with protein G beads for 1 hr. Beads were washed 4 times with modified RIPA and proteins eluted with 6X reducing sample buffer.
Population Doubling Studies, FACS, and Microtubule Regrowth Assays
Population doubling: Cell lines were grown in T25 flasks, counted and replated every 3 days.
Cells from a confluent T75 flask were collected and stained with propidium iodide. Samples were analyzed on a BD FACsort (Beckton Dickinson), 40,000 events collected per sample. FlowJo software (TreeStar) was used to perform cell cycle analysis. Microtubule regrowth assays: Cells were plated on coated coverslips and treated with 1 μM nocodazole for 1 hr in culture to depolymerize microtubules. Cells were fixed with 100% methanol at 0″, 30″, 1′, 2′, 10′, and 15′ after nocodazole washout, and processed for IF as described above.
Cells were plated on 8 well Permanox slides (Nunc), fixed in 3% glutaraldehyde in 0.1M phosphate buffer (PB) for 30′ at room temperature, then washed 3 times in 0.1M PB. Cells were postfixed in 2% osmium for 2 hr, dehydrated and embedded in Araldite (Durcupan, Fluka). Serial ultrathin sections (70nm) were cut with a diamond knife, stained with lead citrate and examined under a FEI Tecnai Spirit electron microscope. Percent of Ofd1Gt cells with long centrioles was determined using information from centrioles in both longitudinal and transverse sections. Quantitative centriole length measurements were performed on longitudinal sections only using ImageTool software.
Centriole Dynamics and Cell Cycle Studies
Cells were plated on coated coverslips, then treated with 10 μg/mL nocodazole for 1 hr in culture, or 0.5 mM mimosine, 3.2 μg/mL aphidocolin, or 2 μM camptothecin (Sigma) overnight. Cells were fixed in 100% methanol and processed for IF as described above.
All error bars represent one standard deviation. For immunofluorescene quantifications, at least 200 cells were counted on each of duplicate coverslips in at least two separate experiments. Student’s unpaired t-test was used to determine statistical significance with a p value of less than 0.05.