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opnc (New England Biolabs)

Name: opnc
Brand: New England Biolabs
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New England Biolabs opnc

A. SPP1 is located in chromosome 4q22.1, spanning 7.76 kb from 87.9756 to 87.9834 Mb in the Contig NC_000004.12. Five isoforms are banked in genome build GRCh38. OPN5 is the only isoform that has exon 4 and an alternative start codon. OPNb lacks exon 6 while <t>OPNc</t> lacks exon 5. OPN4 is the isoform with the shortest transcript, lacking exons 4, 5 and 6. B. Heat map of exome variant analysis using ST 2.1 Affymetrix arrays of 124 primary EACs (exon 6 was not available in the ST 2.1 array). C. Summary of OPN isoform-specific exon expression (0, absence of exon/exons; 1, presence of exon/exons; Frequency, total number of times the exon/exons is expressed across all isoforms). D. Correlation of OPN isoform-specific expression in primary EAC. The mean expression derived from 3 probe sets (2 from exon 7 and 1 from exon 8) with the least deviation among all common exons (1, 2, 3, 7 and 8) was used to represent the total OPN expression. Subtraction of exon 5 expression (specific for <t>isoforms</t> <t>OPNa,</t> b and 5) from the total OPN expression yielded OPNc+4 expression (red dots). Subtraction of exon 4 expression, which was specific for OPN5 (green dots), from exon 5 expression yielded OPNa+b expression (blue dots). These expression levels were then plotted against the total OPN expression for each primary EAC in the arrays. E. Pearson correlation coefficients showed significant correlation between these groups.

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1) Product Images from "Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma"

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

Journal: Oncotarget

doi:

Figure Legend Snippet: A. SPP1 is located in chromosome 4q22.1, spanning 7.76 kb from 87.9756 to 87.9834 Mb in the Contig NC_000004.12. Five isoforms are banked in genome build GRCh38. OPN5 is the only isoform that has exon 4 and an alternative start codon. OPNb lacks exon 6 while OPNc lacks exon 5. OPN4 is the isoform with the shortest transcript, lacking exons 4, 5 and 6. B. Heat map of exome variant analysis using ST 2.1 Affymetrix arrays of 124 primary EACs (exon 6 was not available in the ST 2.1 array). C. Summary of OPN isoform-specific exon expression (0, absence of exon/exons; 1, presence of exon/exons; Frequency, total number of times the exon/exons is expressed across all isoforms). D. Correlation of OPN isoform-specific expression in primary EAC. The mean expression derived from 3 probe sets (2 from exon 7 and 1 from exon 8) with the least deviation among all common exons (1, 2, 3, 7 and 8) was used to represent the total OPN expression. Subtraction of exon 5 expression (specific for isoforms OPNa, b and 5) from the total OPN expression yielded OPNc+4 expression (red dots). Subtraction of exon 4 expression, which was specific for OPN5 (green dots), from exon 5 expression yielded OPNa+b expression (blue dots). These expression levels were then plotted against the total OPN expression for each primary EAC in the arrays. E. Pearson correlation coefficients showed significant correlation between these groups.

Techniques Used: Variant Assay, Expressing, Derivative Assay

A. OPNb cells showed significantly more invasion than OPNc cells using Matrigel Basement Membrane Matrix-casted culture dishes (250 μg/ml of BD Matrigel Matrix) following 24–36 h incubation and Diff-Quick staining as compared with cells in non-casted culture dishes. OPN isoform expression levels were monitored using qRT-PCR. B. Matrigel Matrix-resuspended OPNb cells displayed more invasive growth and xenograft formation in vivo than OPNc cells. One million Lenti-Luc-labeled OE33/OPN stable cells were resuspended in 0.1 ml Matrigel Matrix and subcutaneously injected into the flanks of nude mice. In vivo tumor imaging to monitor growth was performed using a Xenogen IVIS Spectrum scanner (*Note, red asterisk, actual imaging intensity should be greater than the reported measurement (total flux, p/s) due to tumor ulceration; blue asterisk, nodule at the site of subcutaneous injection but luciferin signal not detected).

Figure Legend Snippet: A. OPNb cells showed significantly more invasion than OPNc cells using Matrigel Basement Membrane Matrix-casted culture dishes (250 μg/ml of BD Matrigel Matrix) following 24–36 h incubation and Diff-Quick staining as compared with cells in non-casted culture dishes. OPN isoform expression levels were monitored using qRT-PCR. B. Matrigel Matrix-resuspended OPNb cells displayed more invasive growth and xenograft formation in vivo than OPNc cells. One million Lenti-Luc-labeled OE33/OPN stable cells were resuspended in 0.1 ml Matrigel Matrix and subcutaneously injected into the flanks of nude mice. In vivo tumor imaging to monitor growth was performed using a Xenogen IVIS Spectrum scanner (*Note, red asterisk, actual imaging intensity should be greater than the reported measurement (total flux, p/s) due to tumor ulceration; blue asterisk, nodule at the site of subcutaneous injection but luciferin signal not detected).

Techniques Used: Incubation, Diff-Quik, Staining, Expressing, Quantitative RT-PCR, In Vivo, Labeling, Injection, Imaging

Flo/OPN stable cells were seeded at 0.6 × 10 6 cells/12-well, wounded and cultured in media in the presence or absence of cilengitide (100 nM) up to 96 h post wounding. A. In the absence of cilengitide, both OPNb and OPNib cells showed significantly increased migration as compared to LacZ control cells. Both OPNc and OPNic cells were significantly less motile, with gaps remaining more than 96 h post wounding. OPNa cells showed increased migration as compared to LacZ control cells whereas OPNia cells demonstrated similar migration to LacZ cells. B. In the presence of cilengitide, OPNb cell motility was attenuated while OPNib cell migration was severely hindered. Reduced migration of OPNc and OPNic cells remained unchanged. Increased motility of OPNa cells was also attenuated but OPNia cell migration was similar to LacZ cells (* P < 0.05, *** P < 0.001, **** P < 0.0001, t -test).

Figure Legend Snippet: Flo/OPN stable cells were seeded at 0.6 × 10 6 cells/12-well, wounded and cultured in media in the presence or absence of cilengitide (100 nM) up to 96 h post wounding. A. In the absence of cilengitide, both OPNb and OPNib cells showed significantly increased migration as compared to LacZ control cells. Both OPNc and OPNic cells were significantly less motile, with gaps remaining more than 96 h post wounding. OPNa cells showed increased migration as compared to LacZ control cells whereas OPNia cells demonstrated similar migration to LacZ cells. B. In the presence of cilengitide, OPNb cell motility was attenuated while OPNib cell migration was severely hindered. Reduced migration of OPNc and OPNic cells remained unchanged. Increased motility of OPNa cells was also attenuated but OPNia cell migration was similar to LacZ cells (* P < 0.05, *** P < 0.001, **** P < 0.0001, t -test).

Techniques Used: Cell Culture, Migration

Culture plates were coated with Matrigel mixture (laminin at 10.8 μg/ml), blocked for 30 min, seeded with a single cell suspension (0.01 × 10 6 cells/96-well) and incubated at 37°C/5% CO 2 for 30 min. Cells were then fixed with glutaraldehyde and stained with Diff/Quick solution. A. Representative images demonstrating that OPNb significantly enhanced cell adhesion in laminin casted culture plates as compared to the LacZ control and OPNc stable cells. B. Enhanced cell adhesion was also observed for OPNib- but not OPNic-expressing cells. The significant increase in OPNb cell adhesion was abrogated with the addition of 1000 nM cilengitide. (FOV, field of view)

Figure Legend Snippet: Culture plates were coated with Matrigel mixture (laminin at 10.8 μg/ml), blocked for 30 min, seeded with a single cell suspension (0.01 × 10 6 cells/96-well) and incubated at 37°C/5% CO 2 for 30 min. Cells were then fixed with glutaraldehyde and stained with Diff/Quick solution. A. Representative images demonstrating that OPNb significantly enhanced cell adhesion in laminin casted culture plates as compared to the LacZ control and OPNc stable cells. B. Enhanced cell adhesion was also observed for OPNib- but not OPNic-expressing cells. The significant increase in OPNb cell adhesion was abrogated with the addition of 1000 nM cilengitide. (FOV, field of view)

Techniques Used: Incubation, Staining, Diff-Quik, Expressing

A. Significant cell detachment was observed for OE33/OPNc-expressing cells. OE33/OPN stable cells were seeded in culture dishes at 80% confluence (0.22 × 10 6 cells/ml in 6-well plates) for 24 h, transfected with lipofectamine alone (mock) or siNonTarget (siNT) or untreated for 96 h and then fixed and stained with Diff-Quik staining solution. While OPNb-expressing cells demonstrated adherence to culture dishes, OPNc cells were significantly detached, as exhibited by markedly increased non-stained areas in culture dishes (see also ). B. OE33 cells harbor both MET and ERBB2 gene amplification with corresponding gene overexpression (see ). Silencing of MET using siRNA enhanced OE33/OPNc cell detachment while knockdown of ERBB2 did not alter its existing phenotype. Expression of both OPNa and b in OE33 cells appeared to increase adhesion of these cells, as control LacZ cells exhibited increased detachment following transfection with 10 nM siMET or co-transfection with 5 nM each siMET and siERBB2 as compared to transfection with 10 nM siERBB2 alone. C. Silencing of MET resulted in significant detachment of OPNia-expressing cells but did not change the phenotypes of OPNib or OPNic-expressing cells as compared to their full-length OPN counterparts treated with siMET .

Figure Legend Snippet: A. Significant cell detachment was observed for OE33/OPNc-expressing cells. OE33/OPN stable cells were seeded in culture dishes at 80% confluence (0.22 × 10 6 cells/ml in 6-well plates) for 24 h, transfected with lipofectamine alone (mock) or siNonTarget (siNT) or untreated for 96 h and then fixed and stained with Diff-Quik staining solution. While OPNb-expressing cells demonstrated adherence to culture dishes, OPNc cells were significantly detached, as exhibited by markedly increased non-stained areas in culture dishes (see also ). B. OE33 cells harbor both MET and ERBB2 gene amplification with corresponding gene overexpression (see ). Silencing of MET using siRNA enhanced OE33/OPNc cell detachment while knockdown of ERBB2 did not alter its existing phenotype. Expression of both OPNa and b in OE33 cells appeared to increase adhesion of these cells, as control LacZ cells exhibited increased detachment following transfection with 10 nM siMET or co-transfection with 5 nM each siMET and siERBB2 as compared to transfection with 10 nM siERBB2 alone. C. Silencing of MET resulted in significant detachment of OPNia-expressing cells but did not change the phenotypes of OPNib or OPNic-expressing cells as compared to their full-length OPN counterparts treated with siMET .

Techniques Used: Expressing, Transfection, Staining, Diff-Quik, Amplification, Over Expression, Cotransfection

A. Cilengitide reduces OPNb but not OPNc cell proliferation in 2-D cultures. Representative plates were shown. OPNb, OPNib, OPNc, OPNic stable and LacZ control cells were seeded in sextuplicate at 150 cells/6-well. Sets of 3 wells were mock-treated or treated with IC 50LacZ dose of cilengitide (1000 nM) for 12 days, and clonal foci were fixed and stained with Diff-Quik Staining solution. B. Bar graph representation of stained foci showing significantly reduced OPNb and OPNib cell proliferation in the presence of cilengitide as compared to their untreated cells, which exceeded the 50% (IC 50LacZ ) reduction observed in treated LacZ control, OPNc or OPNic cells. (*** P < 0.001, **** P < 0.0001, t -test) C. Ectopic expression of both OPNb and OPNc enhanced EAC cell colony formation in 3-D soft agar cultures. Integrin inhibition did not significantly alter the colony formation of these cells. Flo/OPN and LacZ control cells (7000 cells/12-well, from the same cell-suspensions in Figure ) were mixed with top-agar media in the presence or absence of cilengitide (1000 nM) and were pipetted onto the casted base-agar with or without cilengitide. Cells were allowed to grow for 20 days with periodic replacement of the overlaid media with or without cilengitide. Agar plates were stained with 0.05% crystal violet in 50% ethanol and imaged using the Leica MXFL III Stereo microscope; Olympus DP-70 digital camera at × 0.8 magnification. Soft agar assays were performed in triplicate. D. Significantly increased colony-formation was observed in Flo/OPNb, OPNib, OPNc and OPNic cells as compared to Flo/LacZ controls and was not altered with the addition of cilengitide. Colonies were counted using ImageJ software, and graphs were generated using Prism software. (red, comparison to untreated LacZ; green, comparison to cilengitide-treated LacZ; ns, not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA)

Figure Legend Snippet: A. Cilengitide reduces OPNb but not OPNc cell proliferation in 2-D cultures. Representative plates were shown. OPNb, OPNib, OPNc, OPNic stable and LacZ control cells were seeded in sextuplicate at 150 cells/6-well. Sets of 3 wells were mock-treated or treated with IC 50LacZ dose of cilengitide (1000 nM) for 12 days, and clonal foci were fixed and stained with Diff-Quik Staining solution. B. Bar graph representation of stained foci showing significantly reduced OPNb and OPNib cell proliferation in the presence of cilengitide as compared to their untreated cells, which exceeded the 50% (IC 50LacZ ) reduction observed in treated LacZ control, OPNc or OPNic cells. (*** P < 0.001, **** P < 0.0001, t -test) C. Ectopic expression of both OPNb and OPNc enhanced EAC cell colony formation in 3-D soft agar cultures. Integrin inhibition did not significantly alter the colony formation of these cells. Flo/OPN and LacZ control cells (7000 cells/12-well, from the same cell-suspensions in Figure ) were mixed with top-agar media in the presence or absence of cilengitide (1000 nM) and were pipetted onto the casted base-agar with or without cilengitide. Cells were allowed to grow for 20 days with periodic replacement of the overlaid media with or without cilengitide. Agar plates were stained with 0.05% crystal violet in 50% ethanol and imaged using the Leica MXFL III Stereo microscope; Olympus DP-70 digital camera at × 0.8 magnification. Soft agar assays were performed in triplicate. D. Significantly increased colony-formation was observed in Flo/OPNb, OPNib, OPNc and OPNic cells as compared to Flo/LacZ controls and was not altered with the addition of cilengitide. Colonies were counted using ImageJ software, and graphs were generated using Prism software. (red, comparison to untreated LacZ; green, comparison to cilengitide-treated LacZ; ns, not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA)

Techniques Used: Staining, Diff-Quik, Expressing, Inhibition, Microscopy, Software, Generated

A. Western blot analysis revealed increased E-cadherin expression in OPNa-, OPNb-, OPNib- and OPNic-expressing cells but decreased expression in OPNc-and OPNia-expressing cells as compared to LacZ controls. Vimentin expression was increased in both OPNb- and OPNib-expressing cells but decreased in OPNa-, OPNia-, OPNc-, and OPNic-expressing cells. B. Elevated β-catenin expression was observed in OPNa-, OPNb- and OPNc-expressing cells. Addition of cilengitide did not alter β-catenin expression in any of the OPN isoform-expressing cells but induced E-cadherin expression in OPNc-expressing cells. GAPDH or β-actin was included as a loading control.

Figure Legend Snippet: A. Western blot analysis revealed increased E-cadherin expression in OPNa-, OPNb-, OPNib- and OPNic-expressing cells but decreased expression in OPNc-and OPNia-expressing cells as compared to LacZ controls. Vimentin expression was increased in both OPNb- and OPNib-expressing cells but decreased in OPNa-, OPNia-, OPNc-, and OPNic-expressing cells. B. Elevated β-catenin expression was observed in OPNa-, OPNb- and OPNc-expressing cells. Addition of cilengitide did not alter β-catenin expression in any of the OPN isoform-expressing cells but induced E-cadherin expression in OPNc-expressing cells. GAPDH or β-actin was included as a loading control.

Techniques Used: Western Blot, Expressing

Image Search Results

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: A. SPP1 is located in chromosome 4q22.1, spanning 7.76 kb from 87.9756 to 87.9834 Mb in the Contig NC_000004.12. Five isoforms are banked in genome build GRCh38. OPN5 is the only isoform that has exon 4 and an alternative start codon. OPNb lacks exon 6 while OPNc lacks exon 5. OPN4 is the isoform with the shortest transcript, lacking exons 4, 5 and 6. B. Heat map of exome variant analysis using ST 2.1 Affymetrix arrays of 124 primary EACs (exon 6 was not available in the ST 2.1 array). C. Summary of OPN isoform-specific exon expression (0, absence of exon/exons; 1, presence of exon/exons; Frequency, total number of times the exon/exons is expressed across all isoforms). D. Correlation of OPN isoform-specific expression in primary EAC. The mean expression derived from 3 probe sets (2 from exon 7 and 1 from exon 8) with the least deviation among all common exons (1, 2, 3, 7 and 8) was used to represent the total OPN expression. Subtraction of exon 5 expression (specific for isoforms OPNa, b and 5) from the total OPN expression yielded OPNc+4 expression (red dots). Subtraction of exon 4 expression, which was specific for OPN5 (green dots), from exon 5 expression yielded OPNa+b expression (blue dots). These expression levels were then plotted against the total OPN expression for each primary EAC in the arrays. E. Pearson correlation coefficients showed significant correlation between these groups.

Article Snippet: Full-length OPNa, OPNb and OPNc products were ligated (T4 DNA ligase, New England BioLabs) into pcDNA4 (Invitrogen), and pooled, multi-clonal stable cells for each isoform were then cultured and maintained in selective zeocin-containing media ( ).

Techniques: Variant Assay, Expressing, Derivative Assay

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: A. OPNb cells showed significantly more invasion than OPNc cells using Matrigel Basement Membrane Matrix-casted culture dishes (250 μg/ml of BD Matrigel Matrix) following 24–36 h incubation and Diff-Quick staining as compared with cells in non-casted culture dishes. OPN isoform expression levels were monitored using qRT-PCR. B. Matrigel Matrix-resuspended OPNb cells displayed more invasive growth and xenograft formation in vivo than OPNc cells. One million Lenti-Luc-labeled OE33/OPN stable cells were resuspended in 0.1 ml Matrigel Matrix and subcutaneously injected into the flanks of nude mice. In vivo tumor imaging to monitor growth was performed using a Xenogen IVIS Spectrum scanner (*Note, red asterisk, actual imaging intensity should be greater than the reported measurement (total flux, p/s) due to tumor ulceration; blue asterisk, nodule at the site of subcutaneous injection but luciferin signal not detected).

Techniques: Incubation, Diff-Quik, Staining, Expressing, Quantitative RT-PCR, In Vivo, Labeling, Injection, Imaging

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: Flo/OPN stable cells were seeded at 0.6 × 10 6 cells/12-well, wounded and cultured in media in the presence or absence of cilengitide (100 nM) up to 96 h post wounding. A. In the absence of cilengitide, both OPNb and OPNib cells showed significantly increased migration as compared to LacZ control cells. Both OPNc and OPNic cells were significantly less motile, with gaps remaining more than 96 h post wounding. OPNa cells showed increased migration as compared to LacZ control cells whereas OPNia cells demonstrated similar migration to LacZ cells. B. In the presence of cilengitide, OPNb cell motility was attenuated while OPNib cell migration was severely hindered. Reduced migration of OPNc and OPNic cells remained unchanged. Increased motility of OPNa cells was also attenuated but OPNia cell migration was similar to LacZ cells (* P < 0.05, *** P < 0.001, **** P < 0.0001, t -test).

Techniques: Cell Culture, Migration

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: Culture plates were coated with Matrigel mixture (laminin at 10.8 μg/ml), blocked for 30 min, seeded with a single cell suspension (0.01 × 10 6 cells/96-well) and incubated at 37°C/5% CO 2 for 30 min. Cells were then fixed with glutaraldehyde and stained with Diff/Quick solution. A. Representative images demonstrating that OPNb significantly enhanced cell adhesion in laminin casted culture plates as compared to the LacZ control and OPNc stable cells. B. Enhanced cell adhesion was also observed for OPNib- but not OPNic-expressing cells. The significant increase in OPNb cell adhesion was abrogated with the addition of 1000 nM cilengitide. (FOV, field of view)

Techniques: Incubation, Staining, Diff-Quik, Expressing

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: A. Significant cell detachment was observed for OE33/OPNc-expressing cells. OE33/OPN stable cells were seeded in culture dishes at 80% confluence (0.22 × 10 6 cells/ml in 6-well plates) for 24 h, transfected with lipofectamine alone (mock) or siNonTarget (siNT) or untreated for 96 h and then fixed and stained with Diff-Quik staining solution. While OPNb-expressing cells demonstrated adherence to culture dishes, OPNc cells were significantly detached, as exhibited by markedly increased non-stained areas in culture dishes (see also ). B. OE33 cells harbor both MET and ERBB2 gene amplification with corresponding gene overexpression (see ). Silencing of MET using siRNA enhanced OE33/OPNc cell detachment while knockdown of ERBB2 did not alter its existing phenotype. Expression of both OPNa and b in OE33 cells appeared to increase adhesion of these cells, as control LacZ cells exhibited increased detachment following transfection with 10 nM siMET or co-transfection with 5 nM each siMET and siERBB2 as compared to transfection with 10 nM siERBB2 alone. C. Silencing of MET resulted in significant detachment of OPNia-expressing cells but did not change the phenotypes of OPNib or OPNic-expressing cells as compared to their full-length OPN counterparts treated with siMET .

Techniques: Expressing, Transfection, Staining, Diff-Quik, Amplification, Over Expression, Cotransfection

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: A. Cilengitide reduces OPNb but not OPNc cell proliferation in 2-D cultures. Representative plates were shown. OPNb, OPNib, OPNc, OPNic stable and LacZ control cells were seeded in sextuplicate at 150 cells/6-well. Sets of 3 wells were mock-treated or treated with IC 50LacZ dose of cilengitide (1000 nM) for 12 days, and clonal foci were fixed and stained with Diff-Quik Staining solution. B. Bar graph representation of stained foci showing significantly reduced OPNb and OPNib cell proliferation in the presence of cilengitide as compared to their untreated cells, which exceeded the 50% (IC 50LacZ ) reduction observed in treated LacZ control, OPNc or OPNic cells. (*** P < 0.001, **** P < 0.0001, t -test) C. Ectopic expression of both OPNb and OPNc enhanced EAC cell colony formation in 3-D soft agar cultures. Integrin inhibition did not significantly alter the colony formation of these cells. Flo/OPN and LacZ control cells (7000 cells/12-well, from the same cell-suspensions in Figure ) were mixed with top-agar media in the presence or absence of cilengitide (1000 nM) and were pipetted onto the casted base-agar with or without cilengitide. Cells were allowed to grow for 20 days with periodic replacement of the overlaid media with or without cilengitide. Agar plates were stained with 0.05% crystal violet in 50% ethanol and imaged using the Leica MXFL III Stereo microscope; Olympus DP-70 digital camera at × 0.8 magnification. Soft agar assays were performed in triplicate. D. Significantly increased colony-formation was observed in Flo/OPNb, OPNib, OPNc and OPNic cells as compared to Flo/LacZ controls and was not altered with the addition of cilengitide. Colonies were counted using ImageJ software, and graphs were generated using Prism software. (red, comparison to untreated LacZ; green, comparison to cilengitide-treated LacZ; ns, not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001, One-way ANOVA)

Techniques: Staining, Diff-Quik, Expressing, Inhibition, Microscopy, Software, Generated

Journal: Oncotarget

Article Title: Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

doi:

Figure Lengend Snippet: A. Western blot analysis revealed increased E-cadherin expression in OPNa-, OPNb-, OPNib- and OPNic-expressing cells but decreased expression in OPNc-and OPNia-expressing cells as compared to LacZ controls. Vimentin expression was increased in both OPNb- and OPNib-expressing cells but decreased in OPNa-, OPNia-, OPNc-, and OPNic-expressing cells. B. Elevated β-catenin expression was observed in OPNa-, OPNb- and OPNc-expressing cells. Addition of cilengitide did not alter β-catenin expression in any of the OPN isoform-expressing cells but induced E-cadherin expression in OPNc-expressing cells. GAPDH or β-actin was included as a loading control.

Techniques: Western Blot, Expressing

Primers for PCR-based assays and siRNAs used for transfection

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: Primers for PCR-based assays and siRNAs used for transfection

Article Snippet: A set of minigene reporters for the splicing of OPNb and OPNc was constructed by Genechem Co., Ltd. (Shanghai, China).

Techniques: Sequencing

RUNX2 overexpression enhanced the expression of OPN, especially of splicing isoform c. a Schematic illustration of isoform-specific primers used for the quantitative analyses of OPN-SIs. b TGF-β induction increased the opn transcript levels unproportionally among different splicing isoforms. c TGF-β preferentially promoted OPNc splicing in A549 cells. d Western blot of RUNX2 in A549 cells transfected with an overexpression plasmid. e Overexpression of RUNX2 altered the OPN-SIs and OPNt mRNA levels. f RUNX2 overexpression selectively increased OPNc transcript levels. g Knockdown of RUNX2 expression attenuated TGF-β induced opn expression in A549 cells

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: RUNX2 overexpression enhanced the expression of OPN, especially of splicing isoform c. a Schematic illustration of isoform-specific primers used for the quantitative analyses of OPN-SIs. b TGF-β induction increased the opn transcript levels unproportionally among different splicing isoforms. c TGF-β preferentially promoted OPNc splicing in A549 cells. d Western blot of RUNX2 in A549 cells transfected with an overexpression plasmid. e Overexpression of RUNX2 altered the OPN-SIs and OPNt mRNA levels. f RUNX2 overexpression selectively increased OPNc transcript levels. g Knockdown of RUNX2 expression attenuated TGF-β induced opn expression in A549 cells

Article Snippet: A set of minigene reporters for the splicing of OPNb and OPNc was constructed by Genechem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Knockdown

Knock down of SRSF1 reduced mRNA levels of OPN-SIs. a RNAi of SRSF1 decreased OPNc levels in A549 cells. b Inhibition of OPNb and OPNc splicing from reporter assays following SRSF1 siRNA transfection. c Western blot of SRSF1 in A549 cells transfected with targeted siRNAs

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: Knock down of SRSF1 reduced mRNA levels of OPN-SIs. a RNAi of SRSF1 decreased OPNc levels in A549 cells. b Inhibition of OPNb and OPNc splicing from reporter assays following SRSF1 siRNA transfection. c Western blot of SRSF1 in A549 cells transfected with targeted siRNAs

Article Snippet: A set of minigene reporters for the splicing of OPNb and OPNc was constructed by Genechem Co., Ltd. (Shanghai, China).

Techniques: Knockdown, Inhibition, Transfection, Western Blot

RUNX2 dependent OPNc splicing required normal activities of HDAC1 or HDAC2. a Treatment of A549 cells with HDACs inhibitor TSA suppressed OPNc splicing induced by RUNX2 overexpression. b Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells following TSA treatment. c Inhibition of HDAC1 activity by NaB deprived RUNX2 induced OPNc splicing. d Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells treated with NaB. e Knockdown of HDAC1 or HDAC2, but not HDAC3, decreased RUNX2-induced OPNc splicing. f Western blots of HDAC1, HDAC2 and HDAC3 from A549 cells transfected with the targeted siRNAs

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: RUNX2 dependent OPNc splicing required normal activities of HDAC1 or HDAC2. a Treatment of A549 cells with HDACs inhibitor TSA suppressed OPNc splicing induced by RUNX2 overexpression. b Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells following TSA treatment. c Inhibition of HDAC1 activity by NaB deprived RUNX2 induced OPNc splicing. d Western blot of RUNX2 and OPN in RUNX2 overexpressed A549 cells treated with NaB. e Knockdown of HDAC1 or HDAC2, but not HDAC3, decreased RUNX2-induced OPNc splicing. f Western blots of HDAC1, HDAC2 and HDAC3 from A549 cells transfected with the targeted siRNAs

Article Snippet: A set of minigene reporters for the splicing of OPNb and OPNc was constructed by Genechem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Western Blot, Inhibition, Activity Assay, Knockdown, Transfection

OPNc overexpression was most potent to induce an invasive phenotype in A549 cells among OPN-SIs. a Overexpression of OPNb and OPNc significantly increased the invasiveness of transfected cells. b Overexpression of OPN-SIs promoted cell mobility and migration. c Immunoblots of EMT marker proteins in cells with overexpression of OPN-SIs

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: OPNc overexpression was most potent to induce an invasive phenotype in A549 cells among OPN-SIs. a Overexpression of OPNb and OPNc significantly increased the invasiveness of transfected cells. b Overexpression of OPN-SIs promoted cell mobility and migration. c Immunoblots of EMT marker proteins in cells with overexpression of OPN-SIs

Article Snippet: A set of minigene reporters for the splicing of OPNb and OPNc was constructed by Genechem Co., Ltd. (Shanghai, China).

Techniques: Over Expression, Transfection, Migration, Western Blot, Marker

TGF-β induced OPNc expression enhanced the mobility of SK-MES-1 cells with a dependence to HDACs. a TGF-β treatments for 48 h increased the invasiveness of SK-MES-1 cells. b TGF-β induction increased OPNt and OPN-SIs levels with a preference to OPNc. c TGF-β promoted OPNc splicing with most significance in SK-MES-1 cells. d Knockdown of HDAC1 or HDAC2 significantly decreased the RUNX2-induced OPNc splicing. e The HDAC1 and HDAC2 expression were markedly reduced in the SK-MES-1 cells transfected with the targeted siRNAs. f Overexpression of OPN-SIs significantly promoted the migration of SK-MES-1 cells

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: TGF-β induced OPNc expression enhanced the mobility of SK-MES-1 cells with a dependence to HDACs. a TGF-β treatments for 48 h increased the invasiveness of SK-MES-1 cells. b TGF-β induction increased OPNt and OPN-SIs levels with a preference to OPNc. c TGF-β promoted OPNc splicing with most significance in SK-MES-1 cells. d Knockdown of HDAC1 or HDAC2 significantly decreased the RUNX2-induced OPNc splicing. e The HDAC1 and HDAC2 expression were markedly reduced in the SK-MES-1 cells transfected with the targeted siRNAs. f Overexpression of OPN-SIs significantly promoted the migration of SK-MES-1 cells

Article Snippet: A set of minigene reporters for the splicing of OPNb and OPNc was constructed by Genechem Co., Ltd. (Shanghai, China).

Techniques: Expressing, Knockdown, Transfection, Over Expression, Migration

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: Primers for PCR-based assays and siRNAs used for transfection

Article Snippet: The expression cassettes of OPN-SIs (OPNa, OPNb and OPNc) and RUNX2 were cloned individually into pENTER vectors by Vigene Bioscience Co., Ltd. (Shandong, China) and amplified in bacterial host cells.

Techniques: Sequencing

Upregulation of RUNX2 associated with increased OPN expression during the EMT progression in TGF-β treated A549 cells. a TGF-β treatments for 48 h increased cell migration. b TGF-β treatments induced significant changes in the expression of major EMT marker genes at both mRNA (left) and protein (right) levels. c The mRNA (upper) and protein (lower) expression of OPN and RUNX2 were synchronically increased following TGF-β treatments

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Figure Lengend Snippet: Upregulation of RUNX2 associated with increased OPN expression during the EMT progression in TGF-β treated A549 cells. a TGF-β treatments for 48 h increased cell migration. b TGF-β treatments induced significant changes in the expression of major EMT marker genes at both mRNA (left) and protein (right) levels. c The mRNA (upper) and protein (lower) expression of OPN and RUNX2 were synchronically increased following TGF-β treatments

Techniques: Expressing, Migration, Marker

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Techniques: Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation, Knockdown

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Techniques: Over Expression, Western Blot, Inhibition, Activity Assay, Knockdown, Transfection

Journal: Cancer Cell International

Article Title: Enhanced osteopontin splicing regulated by RUNX2 is HDAC-dependent and induces invasive phenotypes in NSCLC cells

doi: 10.1186/s12935-019-1033-5

Techniques: Expressing, Knockdown, Transfection, Over Expression, Migration

A. Illustration of OPN isoform primary domain structure. Each block corresponds to an exon (numbered). OPNa is full length (top), OPNb lacks exon 5 (middle), and OPNc lacks exon 4 (bottom). Expanded amino acid sequences of exons 4 and 5, absent in OPNc and OPNb, respectively, are included. B. OPN primers were used to measure OPNa (277bp), OPNb (235bp), and OPNc (196bp) mRNA levels in tissue samples from non-ischemic tissues and tissues from PAD patients with critical limb ischemia by RT-PCR (n=3–5). Isoform plasmid DNA controls and beta actin for loading are both shown. C. To investigate if OPN isoforms differentially affect collateral vessel formation in vivo , the hindlimb ischemia was performed on the following groups: WT, OPN −/− , or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. Perfusion was measured by Laser Doppler perfusion imaging (LDPI). Representative LDPI images 14 days post-HLI are shown. D. Ischemic limb (IL) perfusion was quantified and normalized to the contralateral non-ischemic limb (NIL) and compared across groups. * p<0.05, † p<0.001 vs. GFP; d14, n=6. E. To determine if the OPN isoform effects on perfusion translate to increased functional limb use, animals were given free access to a running wheel (d7 post-HLI) and allowed to run for 7 days. Total distance run (meters) by was plotted for all groups as a measure of limb function. *p<0.05, † p<0.001 vs. GFP; n=9–10.

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: A. Illustration of OPN isoform primary domain structure. Each block corresponds to an exon (numbered). OPNa is full length (top), OPNb lacks exon 5 (middle), and OPNc lacks exon 4 (bottom). Expanded amino acid sequences of exons 4 and 5, absent in OPNc and OPNb, respectively, are included. B. OPN primers were used to measure OPNa (277bp), OPNb (235bp), and OPNc (196bp) mRNA levels in tissue samples from non-ischemic tissues and tissues from PAD patients with critical limb ischemia by RT-PCR (n=3–5). Isoform plasmid DNA controls and beta actin for loading are both shown. C. To investigate if OPN isoforms differentially affect collateral vessel formation in vivo , the hindlimb ischemia was performed on the following groups: WT, OPN −/− , or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. Perfusion was measured by Laser Doppler perfusion imaging (LDPI). Representative LDPI images 14 days post-HLI are shown. D. Ischemic limb (IL) perfusion was quantified and normalized to the contralateral non-ischemic limb (NIL) and compared across groups. * p<0.05, † p<0.001 vs. GFP; d14, n=6. E. To determine if the OPN isoform effects on perfusion translate to increased functional limb use, animals were given free access to a running wheel (d7 post-HLI) and allowed to run for 7 days. Total distance run (meters) by was plotted for all groups as a measure of limb function. *p<0.05, † p<0.001 vs. GFP; n=9–10.

Article Snippet: Cells were allowed to migrate for 2 hours in response to the following stimuli diluted in 0.1% FBS-DMEM: 100 ng/mL monocyte chemoattractant protein (MCP)-1, 50 ng/mL of OPNa, OPNb, or OPNc recombinant protein (Origene).

Techniques: Blocking Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, In Vivo, Imaging, Functional Assay

To determine if OPN isoforms differentially affect arteriogenesis, tissue sections from animals 14 days post-HLI treated (trx) with lentivirus (LV) to overexpress OPN isoform a, b, or c were stained with α smooth muscle actin (α-SMA). α-SMA positive vessel numbers and sizes were quantified as a readout for arteriogenesis. A. The number of α-SMA positive vessels was counted across treatment groups and plotted (p = ns). B. α-SMA positive vessel sizes were measured in WT or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. The number of vessels measured within the arteriole (10 – 200 μm 2 ), small artery (200 – 700 μm 2 ) and large artery (1000 – 2500 μm 2 ) size ranges were compared across all animal groups. Data are expressed as % change compared to +LV-GFP (control). *p<0.05 vs. OPNa, † p<0.001 vs. OPNb; n=8–10. C. Representative histology images from 14 days post-HLI stained with α-SMA are shown. α-SMA stain is red and counterstain is violet. Scale bars = 500 μm.

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Osteopontin Isoforms Differentially Promote Arteriogenesis in Response to Ischemia via Macrophage Accumulation and Survival

doi: 10.1038/s41374-018-0094-8

Figure Lengend Snippet: To determine if OPN isoforms differentially affect arteriogenesis, tissue sections from animals 14 days post-HLI treated (trx) with lentivirus (LV) to overexpress OPN isoform a, b, or c were stained with α smooth muscle actin (α-SMA). α-SMA positive vessel numbers and sizes were quantified as a readout for arteriogenesis. A. The number of α-SMA positive vessels was counted across treatment groups and plotted (p = ns). B. α-SMA positive vessel sizes were measured in WT or OPN −/− mice treated (trx) with lentivirus (LV) to overexpress GFP, OPNa, OPNb, or OPNc. The number of vessels measured within the arteriole (10 – 200 μm 2 ), small artery (200 – 700 μm 2 ) and large artery (1000 – 2500 μm 2 ) size ranges were compared across all animal groups. Data are expressed as % change compared to +LV-GFP (control). *p<0.05 vs. OPNa, † p<0.001 vs. OPNb; n=8–10. C. Representative histology images from 14 days post-HLI stained with α-SMA are shown. α-SMA stain is red and counterstain is violet. Scale bars = 500 μm.

Techniques: Staining

Review

Structured Review

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1) Product Images from "Osteopontin (OPN/ SPP1 ) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma"

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