Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Immunofluorescence was used to detect autotaxin (ATX) expression in visceral and parietal tissues from Wt1 cre/+ ; R26R eYFP/+ reporter mice. Cells that express Wt1, a mesothelial marker, will express enhanced yellow fluorescent protein (eYFP) that can be detected with a green fluorescent protein (GFP) antibody (A, D, G). Robust autotaxin expression is evident in visceral mesothelia (A–F). In contrast, the parietal mesothelium that lines the body wall expresses the mesothelial marker (G), but is devoid of autotaxin expression (H). Nuclei are marked with DAPI. Quantitative real-time RT-PCR was used to determine autotaxin transcript levels in visceral (omental) mesothelium, parietal mesothelium, heart, and liver (J). The autotaxin expression in visceral cells was set to 1 and fold changes were calculated for each subsequent tissue type. The asterisk represents a statistically significant difference when compared to the expression level in visceral cells (p<0.05, n = 4). Error bars were calculated using standard error of the mean.

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Immunofluorescence, Expressing, Marker, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Conditioned media was collected from cultured visceral mesothelia, parietal mesothelia, peritoneal mesothelioma (MSTO), and pleural mesothelioma (ROB) cells. Some cultures were treated with the autotaxin inhibitor S32826. Media was assayed for LPA accumulation as a measure of autotaxin activity over a 2 hour period. Visceral mesothelia had significantly higher levels of autotaxin activity compared to parietal cells (A). Furthermore, MSTO and ROB cells had even higher levels of autotaxin activity compared to normal mesothelial cells (B). Normal and pathological cells were responsive to S32826 and displayed decreased levels of autotaxin activity (A–B).

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Cell Culture, Activity Assay

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Visceral mesothelial, parietal mesothelial, pleural mesothelioma (MSTO), and peritoneal mesothelioma (ROB) cells were cultured on transwell filters with and without addition of autotaxin (ATX), the autotaxin inhibitor S32826, Lpar1 inhibitor 1 (2440), or Lpar1 inhibitor 2 (8437). Of all the cells studied, the mesothelioma cells were the most invasive (C, D). Visceral cells (A) invaded more readily than parietal (B), but addition of ATX to parietal cells (F) promoted invasion to visceral-like levels. Treatment with S32826 reduced cell invasion in all groups (I-L). Furthermore, both of the Lpar1 inhibitors were efficient at decreasing cell invasion in mesothelial (M, Q) and mesothelioma cells (O, P, S, T). The percent of cell invasion is quantified in panel U. The percent of untreated visceral cells that migrated was set to 100. Asterisks represent statistically significant differences compared to untreated values in each group (p<0.05, n = 4). Error bars were calculated using standard error of the mean.

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Cell Culture

Journal: PLoS ONE

Article Title: Autotaxin Signaling Governs Phenotypic Heterogeneity in Visceral and Parietal Mesothelia

doi: 10.1371/journal.pone.0069712

Figure Lengend Snippet: Visceral mesothelial, parietal mesothelial, pleural mesothelioma (MSTO), and peritoneal mesothelioma (ROB) cells were plated on fibronectin, cultured with or without the addition of autotaxin, the autotaxin inhibitor S32826, Lpar1 inhibitor 1 (2440), or Lpar1 inhibitor 2 (8437), and subjected to time-lapse imaging in order to visualize their ability to migrate. Panels A-H are compilations of static images taken at 20 minute intervals. False coloration indicates a cell's location at each interval: red 0 min; green 20 min; yellow 40 min; blue 60 min; purple 80 min.

Article Snippet: Visceral mesothelial, parietal mesothelial, MSTO, and ROB cells were cultured in serum free medium for 48 h. Conditioned media from these cells was collected and used in a commercially available autotaxin activity assay (Echelon, K-4100).

Techniques: Cell Culture, Imaging

Journal: Biomedicines

Article Title: Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration

doi: 10.3390/biomedicines9111727

Figure Lengend Snippet: mDCs secrete high levels of Enpp2. ( A ) DC subsets were cultured and Enpp2 expression measured by qRT-PCR. Values were normalized to GAPDH expression. Data are expressed as the mean ± SEM ( n = 5 independent DC preparations) of duplicate experiments. ( B ) Secreted Enpp2 protein in concentrated (20-fold) conditioned low serum medium from DCs was detected by Western blotting. The bar graphs show data derived from densitometric analysis of western blot data. Values are expressed as the mean ± SEM ( n = 3 independent experiments). ( C ) A fluorescence assay of Enpp2 activity. Enzyme activity in conditioned medium was measured for over 2 h. The bar graph shows the Enpp2 activity rates, expressed as the mean ± SEM ( n = 3 independent DC preparations). ** p < 0.01 and *** p < 0.001, compared with immature (im)DCs.

Article Snippet: Cell culture supernatants were collected and tested in a commercial Enpp2 activity assay (Echelon, K-4100).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Fluorescence, Activity Assay

Journal: Biomedicines

Article Title: Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration

doi: 10.3390/biomedicines9111727

Figure Lengend Snippet: Enpp2 knockdown in mDCs affects their immunogenicity. ( A ) Expression of Enpp2 mRNA in DC subsets (scramble siRNA-treated mDCs; SC, and Enpp2 siRNA-treated mDCs; siEnpp2). Enpp2 expression was measured by qRT-PCR. Values were normalized against GAPDH expression. The bar graph shows the Enpp2 expression relative to levels in imDCs. Data are presented as mean ± SEM ( n = 5 independent DC preparations). ( B ) Levels of secreted Enpp2 protein in concentrated (20-fold) conditioned low serum medium from SC or siEnpp2 were detected by Western blotting. Data in the bar graphs were obtained by densitometric analysis of Western blots. All data are expressed as the mean ± SEM ( n = 3 independent experiments). ( C ) Fluorescence assay of Enpp2 activity. Enzyme activity in conditioned medium was measured for over 2 h. The bar graph shows the Enpp2 activity rates, expressed as the mean ± SEM ( n = 3 independent DC preparations). ( D ) SC and siEnpp2 were stained with the indicated fluorescent-labeled antibodies and analyzed by flow cytometry. The bar graphs show the mean fluorescence intensity (MFI). ( E ) Pro-inflammatory cytokines in culture supernatants from DCs were analyzed by ELISA. Data are expressed as the mean ± SEM ( n = 5 independent DC preparations) of duplicate experiments. ( F ) Each DC subset was co-cultured for 72 h with CD3 + T cells at a ratio of 1:10. The CD3 + T-cells isolated from splenocytes of naïve C57BL/6 mice were stained with CFSE and co-cultured with DCs. ( G ) Cytokine levels in the supernatants after 72 h of co-culture were measured by ELISA. Data are expressed as mean ± SEM ( n = 3 independent co-culture preparations) of duplicate experiments. ( H ) Migration assay. The number of migrating DCs harvested from the lower Transwell chamber was counted by flow cytometry. The bar graph shows the events/minute, expressed as the mean ± SEM ( n = 3 independent DC preparations). ( I ) To detect the indicated proteins, DC lysates were subjected to SDS-PAGE and immunoblotting. Data shown are representatives of three independent experiments. Western blot analysis of RhoA, p38, Erk1/2 expression. ( J ) Fluorescence assay of Enpp2 activity. Enzyme activity in conditioned medium was measured for over 2 h. The bar graph shows the Enpp2 activity rates, expressed as the mean ± SEM ( n = 3 independent DC preparations). ( K ) To detect the indicated proteins, DC lysates were subjected to SDS-PAGE and immunoblotting. Data shown are representatives of three independent experiments. Western blot analysis of RhoA, p38, Erk1/2 expression. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with SC.

Article Snippet: Cell culture supernatants were collected and tested in a commercial Enpp2 activity assay (Echelon, K-4100).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Fluorescence, Activity Assay, Staining, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Isolation, Co-Culture Assay, Migration, SDS Page

Journal: Biomedicines

Article Title: Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration

doi: 10.3390/biomedicines9111727

Figure Lengend Snippet: Enpp2 increases the immunogenicity of mDCs. ( A ) Fluorescence assay of Enpp2 activity. Enzyme activity in conditioned medium was measured over 2 h. The bar graph shows the Enpp2 activity rates, expressed as the mean ± SEM ( n = 3 independent DC preparations). ( B ) DC subsets (mDC; Enpp2- and recombinant Enpp2 protein treated mDCs; Enpp2+) were stained with the indicated fluorescent-labeled antibodies and analyzed by flow cytometry. The bar graphs show the MFI, expressed as the mean ± SEM ( n = 3 independent DC preparations). ( C ) Pro-inflammatory cytokines in culture supernatants from DCs were analyzed by ELISA. Data are expressed as the mean ± SEM ( n = 3 independent DC preparations) of duplicate experiments. ( D ) Each DC subset was co-cultured for 72 h with CFSE-labeled CD3 + T-cells to measure T-cell proliferation. ( E ) Cytokine levels in the supernatants after 72 h of co-culture were measured by ELISA. Data are expressed as mean ± SEM ( n = 3 independent co-culture preparations) of duplicate experiments. ( F ) Migration assay. The number of migrating DCs harvested from the lower Transwell chamber was counted by flow cytometry. The bar graph shows the events/minute, expressed as the mean ± SEM ( n = 3 independent DC preparations). ( G ) Western blot analysis of RhoA, p38, Erk1/2 expression. Data are representative of three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with Enpp2-.

Article Snippet: Cell culture supernatants were collected and tested in a commercial Enpp2 activity assay (Echelon, K-4100).

Techniques: Fluorescence, Activity Assay, Recombinant, Staining, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Co-Culture Assay, Migration, Western Blot, Expressing

Journal: Biomedicines

Article Title: Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration

doi: 10.3390/biomedicines9111727

Figure Lengend Snippet: Enpp2 increases the homing ability of mDCs in vivo. DCs were injected subcutaneously into the right hind leg footpad of C57BL/6 mice, and imaged at 24, 48, and 72. ( A ) DCs (5 × 10 4 cells/50 μL) were injected subcutaneously into mouse ( n = 5 mice for each group) footpads to monitor cell migration to the popliteal lymph nodes. Imaging was performed using the near-infrared 800 nm channel, exciting at a wavelength of 778 nm and detecting at 794 nm. Representative optical images acquired on day 3 post-injection. ( B ) The bar graphs show that fluorescence intensity of DCs migrated to the popliteal lymph nodes (total fluorescence intensity is shown for comparison). Data are expressed as the mean ± SEM ( n = 5 independent mice). ( C ) CFSE (green)-labeled DCs were injected subcutaneously into mouse ( n = 3 mice per group) footpads and confocal micrographs of the popliteal lymph nodes were acquired. Nuclei were stained with DAPI (blue). All images were taken using the same microscope settings and representative images are shown. The bar graphs show the CFSE/DAPI intensity. Scale bar, 100 μm. ( D ) Cells were isolated from the popliteal lymph nodes of each group of mice and then stained with an anti-CD11c antibody prior to flow cytometry analysis. The percentage of CFSE-labeled DCs is indicated on the plots. Data are representative of three independent experiments. * p < 0.05 and ** p < 0.01 compared with Enpp2-.

Article Snippet: Cell culture supernatants were collected and tested in a commercial Enpp2 activity assay (Echelon, K-4100).

Techniques: In Vivo, Injection, Migration, Imaging, Fluorescence, Labeling, Staining, Microscopy, Isolation, Flow Cytometry

Journal: Biomedicines

Article Title: Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration

doi: 10.3390/biomedicines9111727

Figure Lengend Snippet: Human mDCs secrete high levels of Enpp2. ( A ) Human DC subsets (imDC and mDC) were cultured, and Enpp2 expression measured by qRT-PCR. Values were normalized to GAPDH expression. Data are expressed as the mean ± SEM ( n = 5 independent DC preparations) of duplicate experiments. ( B ) Levels of secreted Enpp2 protein in concentrated (20-fold) conditioned serum free media (SFM) from DCs was detected by western blotting. The bar graphs show data derived from densitometric analysis of western blot data. Values are expressed as the mean ± SEM ( n = 3 independent experiments). *** p < 0.001 compared with imDC.

Article Snippet: Cell culture supernatants were collected and tested in a commercial Enpp2 activity assay (Echelon, K-4100).

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay