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Horizon Discovery v atpase
Intracellular localization of subunit <t>a3</t> and markers for the Golgi apparatus and lysosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to the Golgi and lysosomes, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the <t>V-ATPase</t> and Giantin or LAMP2 as markers for the Golgi and lysosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the Golgi apparatus marker Giantin ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the lysosomal marker LAMP2 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.
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1) Product Images from "The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer"

Article Title: The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.10063

Intracellular localization of subunit a3 and markers for the Golgi apparatus and lysosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to the Golgi and lysosomes, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Giantin or LAMP2 as markers for the Golgi and lysosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the Golgi apparatus marker Giantin ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the lysosomal marker LAMP2 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.
Figure Legend Snippet: Intracellular localization of subunit a3 and markers for the Golgi apparatus and lysosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to the Golgi and lysosomes, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Giantin or LAMP2 as markers for the Golgi and lysosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the Golgi apparatus marker Giantin ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the lysosomal marker LAMP2 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.

Techniques Used: Incubation, Staining, Marker, Labeling

Subunit a3 colocalizes with a leading edge marker in migrating breast cancer cells MCF10CA1a, MB231, and SUM149 breast cancer cells were grown to confluence on poly-d-lysine coated coverslips. A wound was made in the cell monolayer and cells were allowed to migrate for 4 h in order to induce the formation of a leading edge. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and the leading edge marker cortactin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. Shown are representative images of MCF10CA1a, MB231, and SUM149 cells stained for cortactin ( left ), a3 ( second from left ), and DAPI ( second from right ). The merged images are present to the far right. White arrows indicate subunit a3 and cortactin colocalization at the leading edge. The images depict representative staining from a minimum of two separate experiments, with 50-80 cells imaged per experiment for each condition tested.
Figure Legend Snippet: Subunit a3 colocalizes with a leading edge marker in migrating breast cancer cells MCF10CA1a, MB231, and SUM149 breast cancer cells were grown to confluence on poly-d-lysine coated coverslips. A wound was made in the cell monolayer and cells were allowed to migrate for 4 h in order to induce the formation of a leading edge. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and the leading edge marker cortactin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. Shown are representative images of MCF10CA1a, MB231, and SUM149 cells stained for cortactin ( left ), a3 ( second from left ), and DAPI ( second from right ). The merged images are present to the far right. White arrows indicate subunit a3 and cortactin colocalization at the leading edge. The images depict representative staining from a minimum of two separate experiments, with 50-80 cells imaged per experiment for each condition tested.

Techniques Used: Marker, Incubation, Staining

Intracellular localization of subunit a3 and markers for early and late endosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to endosomal compartments, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and EEA1 or Rab7 as markers for early endosomes and late endosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the early endosome marker EEA1 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the late endosome marker Rab7 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.
Figure Legend Snippet: Intracellular localization of subunit a3 and markers for early and late endosomes in breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown on poly-d-lysine coated coverslips. To assess whether a3 localizes to endosomal compartments, cells were fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and EEA1 or Rab7 as markers for early endosomes and late endosomes, respectively. Cells were then incubated with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Representative images of each of the cell lines stained for the early endosome marker EEA1 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the inset of the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). B. Representative images of each of the cell lines stained for the late endosome marker Rab7 ( left ), subunit a3 ( second from left ), the merged images ( second from right ), and the merged image magnified 3.5X ( labeled ‘Zoom’, far right ). Colocalization is indicated by the presence of yellow pixels. The images in A and B depict representative staining from a minimum of two separate experiments, with an average of 50 cells imaged per experiment for each condition tested.

Techniques Used: Incubation, Staining, Marker, Labeling

Subunit a3 localizes to the plasma membrane of human breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown to confluence on poly-d-lysine coated coverslips. To assess the effects of cell migration on a3 localization, a wound was made in the cell monolayer and cells were allowed to migrate for 4 h. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Alexa Fluor® 568 phalloidin to stain actin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Shown are representative images of confluent MCF10a, MCF10CA1a, MB231, and SUM149 cells stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). B. Shown are representative images of MCF10a, MCF10CA1a, MB231, and SUM149 cells (with monolayer wounded) stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). White arrows indicate plasma membrane subunit a3. C. Quantification of plasma membrane staining for subunit a3 in confluent or migrating cells. To quantitate plasma membrane staining, 50-80 cells from three separate experiments were counted and the number of cells showing plasma membrane a3 localization was determined. The graphed data represents the percentage of cells showing a3 localization to the plasma membrane. All error bars indicate S.E. *, p
Figure Legend Snippet: Subunit a3 localizes to the plasma membrane of human breast cancer cells MCF10a, MCF10CA1a, MB231, and SUM149 cells were grown to confluence on poly-d-lysine coated coverslips. To assess the effects of cell migration on a3 localization, a wound was made in the cell monolayer and cells were allowed to migrate for 4 h. Cells were then fixed, permeabilized, and immunostained using antibodies against subunit a3 of the V-ATPase and Alexa Fluor® 568 phalloidin to stain actin followed by incubation with secondary antibodies as described under Materials and Methods. Images were taken with identical exposure times and antibody concentrations. A. Shown are representative images of confluent MCF10a, MCF10CA1a, MB231, and SUM149 cells stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). B. Shown are representative images of MCF10a, MCF10CA1a, MB231, and SUM149 cells (with monolayer wounded) stained for phalloidin ( left ), subunit a3 (second from left ), DAPI ( second from right ), and the merged images ( right ). White arrows indicate plasma membrane subunit a3. C. Quantification of plasma membrane staining for subunit a3 in confluent or migrating cells. To quantitate plasma membrane staining, 50-80 cells from three separate experiments were counted and the number of cells showing plasma membrane a3 localization was determined. The graphed data represents the percentage of cells showing a3 localization to the plasma membrane. All error bars indicate S.E. *, p

Techniques Used: Migration, Staining, Incubation