Endogenous OFD1, PC1, PC2, EGFR and flotillin-1 localize to primary cilia of RCTE and MO6-G3 cells.

<p>Polarized RCTE and MO6-G3 cells were fixed and stained using the indicated antibodies. OFD1 from Novus Biologicals (A, F), PC1 using pAb NM002 (B, G), PC2 using AbCam pAb (C, H), EGFR using GeneTex pAb (D, I) and flotillin-1 using AbCam pAb (E, J) were found localized to 100% of primary cilia analyzed for both cell types. Acetylated α-tubulin (Sigma-Aldrich mAb) labeling identifies cilia. Zeiss LSM510 confocal microscope images (63× objective). Arrows denote the protein of interest within a cilium. Representative results from at least 5 independent experiments. Secondary antibody only controls were negative (not shown). Scale bar 10 µm.</p

Expression of PC1, OFD1, EGFR, and Flotillin-1 is decreased in ciliary fractions of PKD cells compared to RCTE cells.

<p>RCTE cells and PKD Q4004X cells were grown 6 days post-confluency to allow for cell polarization and ciliogenesis and treated with 1.5 mM Dibucaine-HCl for 5 minutes to reduce cell loss and induce shedding of primary cilia. Cilia were collected by fractionation and the ciliary fraction was probed. PC1 (MN032 pAb), OFD1 (Novus Biologicals pAb), EGFR (GeneTex pAb), and flotillin-1 (BD Transduction mAb) were expressed in the ciliary fraction of deciliated RCTE cells but not PKD cells. α/β-tubulin (Cell Signaling pAb) was used as a marker of primary cilia. The nuclear marker lamin B (Santa Cruz pAb) was not present in the ciliary fraction (A). Quantification of 3 independent samples (B). Mean values were found to be statistically different (p<0.0001) using 1-way ANOVA.</p

PC1 and EGFR interact in the primary cilium of MO6-G3 and RCTE cells.

<p>Polarized MO6-G3 (A) and RCTE (B) cells were grown on coverslips 5 days post-confluency. Cells were incubated with antibodies against PC1 (Santa Cruz mAb) and EGFR (GeneTex pAb), followed by Duolink PLA Probes to identify points of PC1-EGFR interaction. Cilia identified by acetylated α-tubulin (Sigma-Aldrich mAb). Identical experiments performed without PC1 antibody are negative for fluorescent signal (C). Top panel shows XY plane, bottom panel shows XZ plane. Images from confocal microscope (63× objective). Scale bar is 5 µm. Quantification of puncta in 3 representative images (11–29 cells/field) from 2 independent experiments (D). Quantification of cilia containing PLA puncta (E). Puncta in cilia were counted based on colocalization with acetylated α-tubulin. Statistical evaluation based on two-tailed t-test. p = 0.0194.</p

Key ciliary signaling proteins are significantly reduced in primary cilia of PKD cells.

<p>Human RCTE and PKD Q4004X cells (with expression of mutant PC1) were grown on coverslips 5 days post-confluency to permit ciliogenesis. Cells were fixed and stained using antibodies directed against indicated proteins: PC1 (NM002 pAb); PC2 (AbCam pAb); EGFR (GeneTex pAb); and flotillin-1 (AbCam pAb). PC1 (A), PC2 (C), EGFR (E) and flotillin-1(G) are present in primary cilia of RCTE cells. PC1 (B), PC2 (D), EGFR (F) and flotillin-1 (H) were lacking in primary cilia of PKD Q4004X cells. Acetylated α-tubulin (Sigma-Aldrich mAb) staining identifies cilia. Arrows denote a small residual pool of EGFR detectable at the ciliary base of PKD cells. Zeiss LSM510 confocal microscope images (63× objective). Representative results from at least 5 independent experiments. Comparative images are from a single experiment and taken under identical settings. Arrows denote cilia. Scale bar 10 µm. Quantification shows individual ciliary protein intensities normalized to the respective ciliary volume (I–L). Each protein was quantified in 30–100 cilia for each cell type. z-stack images were imported into SlideBook and a volume mask for each cilium was created based on acetylated α-tubulin staining. Staining intensities for each protein were quantified within the respective ciliary volume mask. Statistical evaluation based on two-tailed t-test. (*) p = 0.0105, (***) p<0.0001.</p

PC1, PC2, EGFR, OFD1, and flotillin-2 are part of a multimeric protein complex in RCTE and MO6-G3 cells.

<p>RCTE and MO6-G3 cells were grown 5 days post-confluency to allow for polarization and ciliogenesis. PC1 was immunoprecipitated using NM002 pAb and precipitated proteins were separated by SDS-PAGE and probed for PC1 using NM005 pAb (A, B), PC2 using AbCam pAb (C), EGFR using GeneTex pAb (D), OFD1 using Novus Biologicals pAb (E) and flotillin-2 using Cell Signaling Rabbit mAb (F). Bar graph showing a densitometric quantification of PC1 co-immunoprecipitation for PC1 (a, b), PC2 (c), EGFR (d), OFD1 (e), and flotillin-2 (f). Normal rabbit IgG was used as a negative control. In a reciprocal experiment EGFR was immunoprecipitated using Santa Cruz pAb from RCTE and MO6-G3 cell lysates. Immunoprecipitated proteins were separated by SDS-PAGE and probed for EGFR using GeneTex pAb (G), PC1 using NM002 pAb (H), PC2 using Santa Cruz pAb (I), OFD1 using Novus Biologicals pAb (J) and flotillin-2 using Cell Signaling Rabbit mAb (K). Bar graph showing a densitometric quantification of EGFR co-immunoprecipitation probed for EGFR (g), PC1 (h), PC2 (i), OFD1 (j), and flotillin-2 (k). Normal rabbit IgG was used as a negative control. Lysate lane inputs were 5% of immunoprecipitations. 25 µg of total protein was loaded into each well. Arrows indicate the quantified band of interest in each immunoblot panel. Bar represents the mean ± SD of at least three independent experiments. (*) p = 0.01 to 0.05, (**) p = 0.001 to 0.01, (***) p<0.001.</p

Key ciliary signaling proteins are expressed in RCTE and MO6-G3 cells.

<p>Renal cortical tubular epithelial (RCTE) cells and tooth derived odontoblasts (MO6-G3) cells were lysed and probed with antibodies directed against indicated proteins. PC1 using NM002 pAb (A, B), PC2 using Santa Cruz pAb (C), OFD1 using Santa Cruz pAb <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106330#pone.0106330-Romio1" target="_blank">[3]</a> (D), EGFR using Santa Cruz pAb (E), ErbB2 using US Biological pAb (F), flotillin-1 using BD Transduction mAb (H), and flotillin-2 using BD Transduction mAb (I) were found to be expressed in both cell types. Actin mAb from Millipore (G) was used as a loading control for PC1, PC2, EGFR, ErbB2, and OFD1; α/β-tubulin pAb from Cell Signaling (J) was used as a loading control for flotillin-1 and flotillin-2. Actin or α/β-tubulin was used to normalize results for quantification. Bar graph showing densitometric quantification of PC1 (a, b), PC2 (c), OFD1 (d), EGFR (e), ErbB2 (f), flotillin-1 (g), flotillin-2 (h). Bar graph represents the mean ± SD of four independent experiments. (*) p = 0.01 to 0.05, (***) p<0.001.</p

Ad5 gene transfer to AI-WAm cells is limited by deficiency in expression and/or cell surface localization of the Ad5 receptor CAR.

<p><b>A.</b> Gene transfer efficiency of a human Ad5 vector expressing Luc reporter (Ad5 (L)) to an AI patient-derived ameloblast-like cells (AI-WAm) at different multiplicities of infection (MOI) (MOI = 10, 50 and 250 TCID₅₀/cell) in comparison to CAR-positive A549 and CAR-negative RD cells by conventional Luc assay at 20 hours post infection. Results are presented in Relative Luc Units (RLU) per cell with mean values shown above each bar plus/minus standard deviation. All differences were statistically significant (<i>P</i><0.05) <b>B.</b> Expression levels of hCAR mRNA in AI-WAm and A549 cells relative to that in RD cells as determined by qRT-PCR and presented as “fold difference”. All differences were statistically significant (P<0.05). <i>P_{(A549/AI-WAm)} = 0.027</i>. <b>C.</b> Quantitative analysis of hCAR and hCD46 mRNA expression levels in RD, AI-WAm and A549 cells as determined by qRT-PCR and normalized to the housekeeping gene <i>GAPDH</i>. The data are presented as ΔΔCt values. For AI-WAm <i>P_(CAR/CD46)</i> = 0.44; for A549 <i>P_(CAR/CD46)</i> = 0.127; <i>for RD P_(CAR/CD46)</i> = 0.007; for CAR <i>P_{(AI-WAm/A549)} = 0.049</i>; <i>P_(RD/AI-WAm) = 0.033</i>; for CD46 <i>P_(RD/AI-WAm) = 0.0011</i>; <i>P_{(AI-WAm/A549)} = 0.0008</i>. <i>P</i>-values for all other differences were <0.05. <b>D.</b> Flow cytometry analysis of Ad5 receptor (CAR) expression in AI-WAm cell population in comparison with CAR-positive (A549) and CAR-negative (RD) control cells. Cells were incubated with primary anti-CAR (RmcB) monoclonal antibody (Ab) followed by labeling with Alexa 488-conjugated secondary antibodies. No primary Ab was used in negative control samples. The extent of shift in the fluorescent peak positions (color lines) relative to control peak(s) of unlabeled cells (black dotted lines) reflects the extent of cell labeling, corresponding to the receptor expression on each cell type and is expressed as Mean Fluorescence Intensity (MFI). Numbers above each peak correspond to percentage (%) of gated (M2) cells calculated using subjective gating. Fluorescence intensity (X-axis) is plotted as histograms on log scale (X-axis) using Flowjo 7.6.4 software (Tree Star Inc., Ashland OR). Y-axis depicts total events (cells) and expressed either as counts or % of maximal. <i>P_{(AI-WAm/A549)} = 0.0038</i>; <i>P_(AI-WAm/RD) = 0.39</i>; <i>P_(A549/RD) = 0.0018</i>; <i>P_{(A549/HEK293T)} = 0.018</i>; CAR <i>P_{(AI-WAm/HEK293)} = 0.0001</i>; <i>P_(HEK293/RD) = 0.001</i>; <b>E.</b> Comparison of hCAR expression in AI-WAm and control cells by IHC staining. CAR-specific (RmcB) primary antibody (same as used for FACS analysis, D) was used to stain AI-WAm cells. A549 and HEK293-T cells were used as positive and RD cells as negative controls for CAR expression. All cells were counter-stained for 5 min. with 300 nM DAPI to visualize nuclear DNA (blue). No primary antibody was used with control samples. Negative control (RD) cells show efficient nuclear staining but no discernible CAR staining (green), while A549 cells demonstrate a strong CAR-specific signal and AI-WAm cells display a moderate level of hCAR signal with diffuse pattern of cytosolic localization (white arrows) similar to that in A549 cells. In sharp contrast, CAR-overexpressing HEK293-T cells show a distinct localization of CAR protein in the cell membrane tight junctions (red arrows) with lesser cytosolic staining. Scale bars correspond to 100 µm.</p

Schematic representation of the Ad5 fiber proteins carrying intact and modified C-terminal knob domains.

<p>Fiber modifications are indicated in the corresponding vector names: Ad5 (G/L) has unmodified fiber knob and possesses the native CAR tropism; Ad5-RGD contains a peptide ligand with an “RGD motif” in the HI loop (red loop) of the fiber knob; Ad5-pK7 contains a stretch of seven lysine residues (green oval) fused to the C-terminus of the Ad5 knob via a (GS)₅ linker (green hook); Ad5-pK7/RGD incorporates both modifications in the corresponding locales of the same fiber molecule; Ad5/3 contains a chimera fiber with Ad5 fiber “knob” domain (gray) replaced with the Ad serotype 3 (Ad3) knob (blue), which retargets the vector to Ad3 receptor(s).</p

Characterization of the human AI-ameloblast cell population by immunohistochemistry, ALP in situ histochemistry and qRT-PCR analysis.

<p><b>A.</b> Image of a tooth extracted from an AI patient that was used to establish an EOE primary cell culture. <b>B.</b> Phase contrast image of AI-WAm cell monolayer. <b>C.</b> AI-WAm cells stained for ALP activity followed by immunostaining for the epithelial marker cytokeratin 14; a single, highly ALP-positive cell is evident (arrow). <b>D.</b> Mouse molar stained for ALP activity showing ALP-negative secretory ameloblasts (Am) with highest activity (dark purple) in the stratum intermedium (SI) followed by the stellate reticulum (SR). Low (<b>E</b>) and high (<b>F</b>) magnification of AI-WAm cells positively stained for the major enamel protein Amel. Arrowheads on panel <b>F</b> indicate cells that appear polarized with unidirectional orientation of the Golgi apparatus. Low (<b>G</b>) and high (<b>H</b>) magnification of AI-WAm cells positively stained for the largest enamel protein Enam. <b>I.</b> Quantitative expression levels of the enamel matrix protein genes <i>AMBN</i>, <i>AMELX</i>, <i>ENAM</i>, <i>AMTN</i> and <i>ODAM</i> in AI-WAm cells relative to dental pulp and normal EOE cells as determined by RT-qPCR following normalization to a housekeeping gene <i>GAPDH</i> and presented as ΔΔCt values. Statistical analysis was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024281#s2" target="_blank">Materials and Methods</a>. Scale bars are: 50 µm (<b>B</b>, <b>F</b>, <b>H</b>), 100 µm (<b>C</b>, <b>D</b>, <b>E</b>, <b>G</b>).</p

The infectivity enhancement effect of fiber-modified Ad5 vectors is mediated by α_vβ3/α_vβ5 integrins and/or HSPG molecules on AI-ameloblasts.

<p><b>A.</b> Differential blocking of gene transfer to AI-WAm cells by integrins. Ad5 RGD shows the highest sensitivity to integrin blocking, while transduction with Ad5-pK7/RGD (G/L) is only partially inhibited. Ad5-pK7 (G/L) gene transfer shows no statistically significant inhibition by integrins. <b>B.</b> Blocking of AI-WAm gene transfer by modified vectors with heparin. Heparin shows a profound dose-dependent blocking effect on transduction with pK7-modified Ads, as opposed to RGD-modified vector. Gray bars (with % values on the top) show percentage of the residual gene transfer level (RLU) resulting from blocking relative to that of unblocked controls (100%) shown by black bar for each fiber-modified vector. All bars represent mean values with standard deviations. All differences were statistically significant except where indicated by asterisk and <i>P</i> values (<i>P</i>>0.05) on the data bars.</p