Expression of receptor transcripts in epithelial cells.

<p><b>A.</b> RT-PCR was performed on HCLE mRNA showing presence of P2Y 1, 2, 4, 6, 11, and 12 receptor transcripts and P2X 4, 5, 6, and 7 receptor transcripts. GAPDH is included as internal control. <b>B.</b> PCR was performed on genomic DNA using exon specific primers. <b>C.</b> RT-PCR was performed on HCLE mRNA using primers that spanned exon 8 (to amplify variants f and j), showing an expected product size of 169 bp. GAPDH was amplified as an internal control. All products were sequenced and verified. Data is representative of a minimum of 3 independent experiments.</p

Cytotoxicity is not induced in epithelial cells following P2X₇ activation.

<p><b>A.</b> MTT assay for cell toxicity was performed on confluent HCLE cells. The response to control media, 100 µM BzATP or actinomycin D is presented as corrected absorbance (570nm–690nm). Data are representative of 3 independent experiments and are presented as +/− SEM. (Significance was determined by ANOVA followed by Tukey posthoc test; ** p<0.001. <b>B.</b> HCLE cells were stimulated with 100 µM BzATP, 2 µg/ml actinomycin D or control media lacking growth factors for 20 hr. Caspase activation was detected with NucView 488 Live Cell Caspase-3 Assay (scale bar  = 50 µm and applies to all 3 images). Images are representative of 5 independent experiments.</p

HCLEs express a full length and truncated P2X₇ mRNA transcript and protein.

<p><b>A.</b> mRNA was purified from total RNA of HCLE and IMR90 cells cultured for 72 hours by twice passing over an oligo-dT column. Purity was confirmed by methylene blue staining of nytran filter following transfer (note lack of rRNA in mRNA lanes). <b>B</b>. Northern blot analysis of HCLE and IMR90 mRNA from 72 hour cultures. The probe used was generated by PCR amplification of a consensus region in all P2X₇ variants. The relative position of 18s rRNA is indicated and the smaller transcript expressed by epithelial cells is indicated (arrow). <b>C.</b> Expression of P2X₇ and P2X_7j mRNA transcript over a 72 hour incubation. Relative expression was determined using the ΔΔCT method. <b>D.</b> Expression of P2X₇ receptor by IMR90 cells. Cells were cultured for 72 hours, lysed and protein was resolved by SDS-PAGE (12%) and immunoblotted with anti-P2X₇. <b>E.</b> Expression of P2X₇ receptor by HCLE cells. Cells were cultured for 72 hours, lysed and protein was resolved by SDS-PAGE (12%) and immunoblotted with anti-P2X₇. Hetero- and homotrimers are identified on crosslinking gels. Epithelial cells were cultured for 72 hours, incubated in situ in the presence of formaldehyde, lysed and incubated at 65°C or 95°C to maintain or break crosslinks. Inset – equivalent experiment resolved by SDS-PAGE (8%) and immunoblotted with anti-P2X₇. Data is representative of a minimum of 3 experiments.</p

Diabetic corneal epithelium displays enhanced P2X₇ receptor expression compared to control non-diabetic corneal epithelium.

<p>Central corneal epithelium from 6 samples of control and diabetic were analyzed using real time PCR. Relative expression of the P2X₇ receptor (all variants), P2X_7j variant receptor, and Ki67 were determined using the ΔΔCT method. The average relative expression of 6 independent samples +/− SEM is presented. Significance was determined by Student's t-test: **p<0.002, *p<0.05 respectively.</p

Expression of P2X₇ receptor changes with stratification.

<p><b>A.</b> Stratification was induced after day 4 (arrow) and cells were cultured for 4 additional days. Cells were lysed and equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-P2X₇. Representative blot is shown. Quantification of 75 kDa and 42 kDa protein are graphed as densitometric values relative to β-actin. Data is representative of a minimum of 4 independent experiments. <b>B.</b> Relative expression of P2X₇ mRNA was determined using the ΔΔCT method and the average of 3 independent cultures +/− SEM is presented. <b>C.</b> Apical cells in stratified culture of HCLE cells at day 7 prestimulated with BzATP show minimal uptake of ToPro-3 using the flow through aparatus. Cultures were prestimulated with BzATP for 5 minutes at which time ToPro-3 in the presence of BzATP was added. Cells were imaged for an additional 20 minutes in the presence of BzATP as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028541#pone-0028541-g004" target="_blank">Figure 4</a> (scale bar  = 50 µm). Arrow indicates representative areas of uptake. Control experiments were performed in the presence of HEPES buffer. <b>D.</b> Relative expression of Ki67 mRNA was determined using the ΔΔCT method and the average of 3 independent cultures +/− SEM is presented.</p

BzATP causes an increase in intracellular Ca²⁺.

<p>HCLE cells were incubated in 5 µM fluo-3AM for 30 min and imaged in a flow-through apparatus on a Zeiss LSM 510 confocal microscope. Cells were washed with indicated HEPES-buffered saline (control, Ca²⁺ free, 4 mM Mg²⁺, 100 µM ZnSO₄) and stimulated with BzATP in corresponding buffer for 2 min. Maximal percent change in average fluorescence of a 460 µm×460 µm field was determined. <b>A.</b> HCLE cells stimulated with BzATP in HEPES buffer, in Ca²⁺ free - HEPES buffer, in HEPES buffer with Mg²⁺ and in HEPES buffer with Zn²⁺. Graphs represent a minimum of six independent experiments +/− SEM. Analysis of variance was determined using the general linear model procedure followed by Tukey-Kramer posthoc test. **p<0.001. <b>B</b>. Response of HCLE cells to ATP followed by stimulation with BzATP. Representative trace of changes in fluorescence over time. Graph represents difference between stimulation with ATP and BzATP after ATP and represents a minimum of six independent experiments +/− SEM. Students t-test ** p<0.001. <b>C</b> Response of HCLE cells to BzATP followed by ATP. Representative trace of changes in fluoresence over time. Graph represents difference between stimulation with BzATP and ATP after BzATP and represents a minimum of six independent experiments +/− SEM. <b>D.</b> Cells were washed with HEPES buffered saline with the indicated concentrations of A438079 and stimulated with BzATP or equimolar concentrations of ADP for 2 min in the presence or absence of inhibitor. The average maximum percent change in fluorescence is graphed as a percentage of the uninhibited control +/− SEM. Each time point is an average of 12 independent experiments. Linear regression analysis where the slope of the line for BzATP is significantly different from 0. p = 0.0001. The control ADP is not significantly different from 0.</p

Confluent monolayer corneal epithelial cells do not form large pores when stimulated with BzATP.

<p>HCLE or IMR90 cells were cultured and stimulated with control media or media containing 100 µM BzATP in the presence of EtBr or ToPro-3 and imaged over 20 min while maintained at 37°C and 5% CO₂ in an environmental chamber on a Zeiss LSM 510 confocal microscope. Representative images are of cells after 20 min BzATP with EtBr (EtBr) or BzATP with ToPro-3 (ToPro)(Scale bar  = 50 µm). Control treated cells in the presence of EtBr are shown (Control). ToPro-3 is detected in IMR90 cells and only detected in HCLE cells being shed. The time to initial EtBr uptake is presented. Images are representative of 3 independent experiments.</p

P2X₇ activation induces phosphorylation of ERK and cell migration.

<p><b>A.</b> Cells were treated in the presence or absence of BzATP for 5 min, lysed and equivalent amounts of protein were resolved by SDS-PAGE and immunoblotted with anti-p-ERK. Blots were stripped and reprobed with anti-ERK and pERK normalized to ERK. Significance was determined by Student's test (p<0.05). Blot is representative of 3 independent experiments. <b>B.</b> Transwell migration assays were performed for 8 hr in the presence of BzATP or binding buffer (control). Migrated cells were stained with propidium iodide, counted in 6 randomly chosen fields (1.48 mm²) and averaged. Data are representative of 3 independent experiments and are presented as mean +/− SEM. Significance was determined by Student's t-test: **p<0.001. <b>C.</b> Downregulation of P2X₇ attenuates wound healing in a representative experiment. HCLE cells transfected with control (non-targeting siRNA) or siRNA targeted to P2X₇ receptor were cultured to confluence. Unsupplemented media or media with 100 µM BzATP were added prior to injury. Slides were placed on a heated stage in an environmental chamber at 37°C and 5% CO₂. Scratch wounds were made and contiguous images were taken every 20 min over 20 hr. Data are representative of 3 independent experiments. <b>D</b>. Percent wound closure from directed migration experiments at endpoint are presented as mean +/− SEM. Significance was determined by ANOVA followed by Tukey posthoc test: **p<0.001. <b>E.</b> HCLE cells transfected with control (non-targeting siRNA) or siRNA targeted to P2X₇ receptor were cultured to confluence. Real time RT-PCR was performed and relative expression of the indicated receptors was determined using the ΔΔCT method. The average relative expression of 3 independent experiments +/− SEM is presented. Significance was determined by Student's test: **p<0.004. <b>F.</b> Parallel experiments to <b>E</b>. were conducted and cultures were lysed and equivalent amounts of protein resolved by SDS-PAGE and immunoblotted with antibodies directed to P2X₇ and P2X₄ receptors.</p

ALI cultured HAECs were infectible by RV-A16.

<p>Immunofluorescence micrographs of transverse sections of ALI cultures shows RV-A16 replicated in ALI cultured HAECs. Positive immunofluorescence staining for mabj2 (in green) was not observed in uninfected control tissues (top panel), but was observed in RV-A16 infected tissues (bottom panel); DAPI staining in blue shows cell nuclei in the tissues. Scale bars: 50 μm.</p

MUC5AC expression is upregulated in response to HRV infection.

<p>A) Immunofluorescence of MUC5AC in ALI cultures from donors 21 (non-asthma, non-smoker), 23 (non-asthma, smoker), 26 (asthma, non-smoker) and 11(asthma, smoker) show an increase in staining with HRV treatment, with only donor 23 having high baseline expression. Green: MUC5AC, blue: DAPI staining for nuclei, representative images, scale bar: 50 μm. N = 6 ALI cultures per group (asthma and non-asthma). B and C) Quantification of immunofluorescence staining presented as either dot plot (B) or box plot (C). Positively stained area was measured and presented as % of total epithelial area. Red dots indicate outliers (> 1.5x inter-quartile distance). B) Inclusion of all data from 6 non-asthmatic and 6 asthmatic donors in analysis. C) Removal of non-diseased smokers (donors 11 and 23) from the data set results in significant differences between HRV and vehicle treated tissues in both non-asthmatic and asthmatic donors as determined by ANOVA and Tukey HSD test.</p