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    Santa Cruz Biotechnology pmca4b shrna plasmid
    <t>PMCA4b</t> but not the trafficking mutant PMCA4b-LA changed shape, culture morphology, and migration-type of A375 cells. (A+B) A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells were cultured in a 6-well plate. After overnight attachment and at 80% confluency images were taken using a phase-contrast microscope. Cell culture morphology was highlighted by applying a black mask to display the contour of the cells. White and yellow arrowheads show protrusions and lamellipodia, respectively. Scale bar, ( A ) 10 µm and ( B ) 50 µm. ( C ) After 48 h in culture, phase-contrast microscopy images were taken. Cell centers were determined and nearest neighbor distances were calculated from the binary images. Insets show the center of cells, as dots. ( D ) Cells were cultured in a 96-well plate and stained with Hoechst 33342. Migratory activity of the cells was followed by recording Hoechst and GFP signals by automated fluorescence microscopy for 24 h. Single cell trajectories of A375-GFP ( n = 130), A375-GFP-PMCA4b ( n = 77), and A375-GFP-PMCA4b-LA ( n = 101) with the starting position of each trajectory translated to the origin of the plot are shown. Mean velocity ± S.D was determined from single cell trajectories (A375-GFP ( n = 645), A375-PMCA4b ( n = 941), and A375-PMCA4b-LA ( n = 990) of 4–5 independent measurements. ( E ) For directional cell migration Boyden chamber assay was performed. Cells were seeded into the upper chamber and left to migrate for 3 h through the filter membrane towards the fibronectin at the lower chamber. Cells at the bottom of the filter membrane were fixed and stained with Toluidine blue. The number of migrated cells from six field of view was counted. Data show the means (% relative to the control) ± S.E.M from three independent experiments (*** p < 0.001).
    Pmca4b Shrna Plasmid, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmca4b shrna plasmid/product/Santa Cruz Biotechnology
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pmca4b shrna plasmid - by Bioz Stars, 2024-04
    92/100 stars
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    91
    Santa Cruz Biotechnology atp2b4 sirna
    <t>PMCA4b</t> but not the trafficking mutant PMCA4b-LA changed shape, culture morphology, and migration-type of A375 cells. (A+B) A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells were cultured in a 6-well plate. After overnight attachment and at 80% confluency images were taken using a phase-contrast microscope. Cell culture morphology was highlighted by applying a black mask to display the contour of the cells. White and yellow arrowheads show protrusions and lamellipodia, respectively. Scale bar, ( A ) 10 µm and ( B ) 50 µm. ( C ) After 48 h in culture, phase-contrast microscopy images were taken. Cell centers were determined and nearest neighbor distances were calculated from the binary images. Insets show the center of cells, as dots. ( D ) Cells were cultured in a 96-well plate and stained with Hoechst 33342. Migratory activity of the cells was followed by recording Hoechst and GFP signals by automated fluorescence microscopy for 24 h. Single cell trajectories of A375-GFP ( n = 130), A375-GFP-PMCA4b ( n = 77), and A375-GFP-PMCA4b-LA ( n = 101) with the starting position of each trajectory translated to the origin of the plot are shown. Mean velocity ± S.D was determined from single cell trajectories (A375-GFP ( n = 645), A375-PMCA4b ( n = 941), and A375-PMCA4b-LA ( n = 990) of 4–5 independent measurements. ( E ) For directional cell migration Boyden chamber assay was performed. Cells were seeded into the upper chamber and left to migrate for 3 h through the filter membrane towards the fibronectin at the lower chamber. Cells at the bottom of the filter membrane were fixed and stained with Toluidine blue. The number of migrated cells from six field of view was counted. Data show the means (% relative to the control) ± S.E.M from three independent experiments (*** p < 0.001).
    Atp2b4 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp2b4 sirna/product/Santa Cruz Biotechnology
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp2b4 sirna - by Bioz Stars, 2024-04
    91/100 stars
      Buy from Supplier

    Image Search Results


    PMCA4b but not the trafficking mutant PMCA4b-LA changed shape, culture morphology, and migration-type of A375 cells. (A+B) A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells were cultured in a 6-well plate. After overnight attachment and at 80% confluency images were taken using a phase-contrast microscope. Cell culture morphology was highlighted by applying a black mask to display the contour of the cells. White and yellow arrowheads show protrusions and lamellipodia, respectively. Scale bar, ( A ) 10 µm and ( B ) 50 µm. ( C ) After 48 h in culture, phase-contrast microscopy images were taken. Cell centers were determined and nearest neighbor distances were calculated from the binary images. Insets show the center of cells, as dots. ( D ) Cells were cultured in a 96-well plate and stained with Hoechst 33342. Migratory activity of the cells was followed by recording Hoechst and GFP signals by automated fluorescence microscopy for 24 h. Single cell trajectories of A375-GFP ( n = 130), A375-GFP-PMCA4b ( n = 77), and A375-GFP-PMCA4b-LA ( n = 101) with the starting position of each trajectory translated to the origin of the plot are shown. Mean velocity ± S.D was determined from single cell trajectories (A375-GFP ( n = 645), A375-PMCA4b ( n = 941), and A375-PMCA4b-LA ( n = 990) of 4–5 independent measurements. ( E ) For directional cell migration Boyden chamber assay was performed. Cells were seeded into the upper chamber and left to migrate for 3 h through the filter membrane towards the fibronectin at the lower chamber. Cells at the bottom of the filter membrane were fixed and stained with Toluidine blue. The number of migrated cells from six field of view was counted. Data show the means (% relative to the control) ± S.E.M from three independent experiments (*** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b but not the trafficking mutant PMCA4b-LA changed shape, culture morphology, and migration-type of A375 cells. (A+B) A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells were cultured in a 6-well plate. After overnight attachment and at 80% confluency images were taken using a phase-contrast microscope. Cell culture morphology was highlighted by applying a black mask to display the contour of the cells. White and yellow arrowheads show protrusions and lamellipodia, respectively. Scale bar, ( A ) 10 µm and ( B ) 50 µm. ( C ) After 48 h in culture, phase-contrast microscopy images were taken. Cell centers were determined and nearest neighbor distances were calculated from the binary images. Insets show the center of cells, as dots. ( D ) Cells were cultured in a 96-well plate and stained with Hoechst 33342. Migratory activity of the cells was followed by recording Hoechst and GFP signals by automated fluorescence microscopy for 24 h. Single cell trajectories of A375-GFP ( n = 130), A375-GFP-PMCA4b ( n = 77), and A375-GFP-PMCA4b-LA ( n = 101) with the starting position of each trajectory translated to the origin of the plot are shown. Mean velocity ± S.D was determined from single cell trajectories (A375-GFP ( n = 645), A375-PMCA4b ( n = 941), and A375-PMCA4b-LA ( n = 990) of 4–5 independent measurements. ( E ) For directional cell migration Boyden chamber assay was performed. Cells were seeded into the upper chamber and left to migrate for 3 h through the filter membrane towards the fibronectin at the lower chamber. Cells at the bottom of the filter membrane were fixed and stained with Toluidine blue. The number of migrated cells from six field of view was counted. Data show the means (% relative to the control) ± S.E.M from three independent experiments (*** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Mutagenesis, Migration, Cell Culture, Microscopy, Staining, Activity Assay, Fluorescence, Boyden Chamber Assay

    PMCA4b but not the trafficking mutant PMCA4b-LA increased cell–cell connections between A375 cells. ( A.1 ) Confocal microscopy images of A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells labeled with Phalloidin-TRITC. Scale bar, 20 µm. Insets show images with higher magnification of field marked with white rectangles; arrowhead indicate cell–cell connection. Scale bar, 5 µm. ( A.2 ) High magnification DIC and fluorescence images of A375-GFP-PMCA4b cells are taken from the field marked with the yellow rectangle in ( A.1 ). Scale bar, 20 µm. The black and red insets show images for the intercellular connections formed between cells. Scale bar, 5 µm ( A.3 ) The graph represents the mean number of connections/cell for 12–13 cell. ( B.1 ) Live cell imaging of A375 cells transiently expressing GFP-actin and mCherry-PMCA4b recorded by spinning-disc confocal microscopy. GFP and mCherry signals were recorded every 0.2 s for 180 s at 37 °C. Scale bar, 20 µm. Insets show the formation of connection between two cells with higher magnification at different times. Scale bar, 5 µm. ( B.2 ) The graph depicts the time courses of GFP-actin and mCherry-PMCA4b signals determined in the region of interest (ROI) drawn around a newly forming connection (yellow polygon). Arrowheads indicate the increased signal (*** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b but not the trafficking mutant PMCA4b-LA increased cell–cell connections between A375 cells. ( A.1 ) Confocal microscopy images of A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells labeled with Phalloidin-TRITC. Scale bar, 20 µm. Insets show images with higher magnification of field marked with white rectangles; arrowhead indicate cell–cell connection. Scale bar, 5 µm. ( A.2 ) High magnification DIC and fluorescence images of A375-GFP-PMCA4b cells are taken from the field marked with the yellow rectangle in ( A.1 ). Scale bar, 20 µm. The black and red insets show images for the intercellular connections formed between cells. Scale bar, 5 µm ( A.3 ) The graph represents the mean number of connections/cell for 12–13 cell. ( B.1 ) Live cell imaging of A375 cells transiently expressing GFP-actin and mCherry-PMCA4b recorded by spinning-disc confocal microscopy. GFP and mCherry signals were recorded every 0.2 s for 180 s at 37 °C. Scale bar, 20 µm. Insets show the formation of connection between two cells with higher magnification at different times. Scale bar, 5 µm. ( B.2 ) The graph depicts the time courses of GFP-actin and mCherry-PMCA4b signals determined in the region of interest (ROI) drawn around a newly forming connection (yellow polygon). Arrowheads indicate the increased signal (*** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Mutagenesis, Confocal Microscopy, Labeling, Fluorescence, Live Cell Imaging, Expressing

    Lamellipodia and stress fiber formation are increased in A375 cells expressing GFP-PMCA4b but not in those expressing GFP-PMCA4b-LA. ( A ) Confocal microscopy images of A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells labeled with Phalloidin-TRITC. Arrowheads show stress fibers. Scale bar, 20 µm. The fractions of stress fiber-positive cells in A375-GFP ( n = 55), A375-GFP-PMCA4b-LA ( n = 72), A375-GFP-PMCA4b ( n = 60) ± PMCA4b siRNA ( n = 68), and negative siRNA ( n = 49) cultures were determined. siRNA confocal microscopy images are presented in . Relative abundance of cells with stress fibers is indicated by the bar graph. Confocal sections were taken from the bottom of the cells to show the stress fibers. ( B ) Confocal microscopy images of A375 and A375-GFP-PMCA4b cells immunostained with vinculin and labeled with Phalloidin-TRITC. Scale bar, 20 µm. Insets show part of the cells with higher magnification. Arrowheads show the differential localization of vinculin. Scale bar, 5 µm. ( C ) A375 and A375-GFP-PMCA4b cells were cultured in a 6-well plate for 48 h. Vinculin protein expression from total cell lysate was analyzed by Western blotting. β-tubulin was used as a loading control. Data represent mean ± S.E.M from three independent experiments (*** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: Lamellipodia and stress fiber formation are increased in A375 cells expressing GFP-PMCA4b but not in those expressing GFP-PMCA4b-LA. ( A ) Confocal microscopy images of A375-GFP, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells labeled with Phalloidin-TRITC. Arrowheads show stress fibers. Scale bar, 20 µm. The fractions of stress fiber-positive cells in A375-GFP ( n = 55), A375-GFP-PMCA4b-LA ( n = 72), A375-GFP-PMCA4b ( n = 60) ± PMCA4b siRNA ( n = 68), and negative siRNA ( n = 49) cultures were determined. siRNA confocal microscopy images are presented in . Relative abundance of cells with stress fibers is indicated by the bar graph. Confocal sections were taken from the bottom of the cells to show the stress fibers. ( B ) Confocal microscopy images of A375 and A375-GFP-PMCA4b cells immunostained with vinculin and labeled with Phalloidin-TRITC. Scale bar, 20 µm. Insets show part of the cells with higher magnification. Arrowheads show the differential localization of vinculin. Scale bar, 5 µm. ( C ) A375 and A375-GFP-PMCA4b cells were cultured in a 6-well plate for 48 h. Vinculin protein expression from total cell lysate was analyzed by Western blotting. β-tubulin was used as a loading control. Data represent mean ± S.E.M from three independent experiments (*** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Confocal Microscopy, Labeling, Cell Culture, Western Blot

    PMCA4b activity is necessary for lamellipodia formation in A375 melanoma cells. ( A ) A375 cells were transfected with GFP-actin together with one of the following plasmids: pmCherry-C1, mCherry-PMCA4b, mCherry-PMCA4b-DE, and cultured for 48 h. Confocal microscopy images of lamellipodia are shown. Scale bar, 20 µm. Graphs represent the GFP-actin intensity profile (green) for the line (white) drawn along the cell as indicated in the confocal image. Confocal sections were taken in the middle to visualize lamellipodia formation. ( B ) The graph shows the fraction of lamellipodia-positive cells in cultures transfected with GFP-actin and mCherry, mCherry-PMCA4b, or mCherry-PMCA4b-DE. ( C , D ) Live-cell imaging of cells expressing GFP-actin and mCherry-PMCA4b. Z-stacks of images were taken every 5 s for 5 min at 37 °C using spinning-disc confocal microscopy. ( C ) Kymographs were generated along the lines (yellow and blue) of the lamellipodia of the mCherry-PMCA4b cell shown in the image in ( A ), using the ImageJ software. Fine edges of ruffles are shown. ( D ) Magnified part of the lamellipodia from the same cell shown in ( A ). White arrowheads show mCherry-PMCA4b positive vesicles and the yellow arrowheads show GFP-actin retrograde flow. Scale bar, 5 µm (*** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b activity is necessary for lamellipodia formation in A375 melanoma cells. ( A ) A375 cells were transfected with GFP-actin together with one of the following plasmids: pmCherry-C1, mCherry-PMCA4b, mCherry-PMCA4b-DE, and cultured for 48 h. Confocal microscopy images of lamellipodia are shown. Scale bar, 20 µm. Graphs represent the GFP-actin intensity profile (green) for the line (white) drawn along the cell as indicated in the confocal image. Confocal sections were taken in the middle to visualize lamellipodia formation. ( B ) The graph shows the fraction of lamellipodia-positive cells in cultures transfected with GFP-actin and mCherry, mCherry-PMCA4b, or mCherry-PMCA4b-DE. ( C , D ) Live-cell imaging of cells expressing GFP-actin and mCherry-PMCA4b. Z-stacks of images were taken every 5 s for 5 min at 37 °C using spinning-disc confocal microscopy. ( C ) Kymographs were generated along the lines (yellow and blue) of the lamellipodia of the mCherry-PMCA4b cell shown in the image in ( A ), using the ImageJ software. Fine edges of ruffles are shown. ( D ) Magnified part of the lamellipodia from the same cell shown in ( A ). White arrowheads show mCherry-PMCA4b positive vesicles and the yellow arrowheads show GFP-actin retrograde flow. Scale bar, 5 µm (*** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Transfection, Cell Culture, Confocal Microscopy, Live Cell Imaging, Expressing, Generated, Software

    PMCA4b silencing in MCF-7 cells changed cell culture morphology and resulted in a dramatic loss of stress fibers. ( A ) Phase contrast images of MCF-7 cells, GFP-PMCA4b (MCF-7-GFP-4b), or shRNA of PMCA4b (MCF-7-Sh-4b). Scale bar, 50 µm. The distance between nearest neighbors was determined using the binary images of cell centers. ( B ) F-actin in the three cell types was labeled with Phalloidin-TRITC. Arrowheads show stress fibers. Scale bar, 20 µm. The fractions of stress fiber-positive cells were determined ( n = 48, 83, and 100, respectively) ( C ) and the area of individual cells of the phase contrast images was calculated ( n = 100, 88, and 70, respectively) ( D ). Bars represent the mean values for each cell type (* p < 0.05, *** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b silencing in MCF-7 cells changed cell culture morphology and resulted in a dramatic loss of stress fibers. ( A ) Phase contrast images of MCF-7 cells, GFP-PMCA4b (MCF-7-GFP-4b), or shRNA of PMCA4b (MCF-7-Sh-4b). Scale bar, 50 µm. The distance between nearest neighbors was determined using the binary images of cell centers. ( B ) F-actin in the three cell types was labeled with Phalloidin-TRITC. Arrowheads show stress fibers. Scale bar, 20 µm. The fractions of stress fiber-positive cells were determined ( n = 48, 83, and 100, respectively) ( C ) and the area of individual cells of the phase contrast images was calculated ( n = 100, 88, and 70, respectively) ( D ). Bars represent the mean values for each cell type (* p < 0.05, *** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Cell Culture, shRNA, Labeling

    PMCA4b expression does not affect F-actin turnover in A375 cells. ( A ) A375 cells were transfected with GFP-actin together with one of the following plasmids, pmCherry-C1, mCherry-PMCA4b, mCherry-PMCA4b-DE, and cultured for 48 h. Media were changed to phenol free DMEM and cells were kept at 37 °C. GFP-actin was photobleached at the ruffles (mCherry: n = 8, mCherry-4b: n = 13, mCherry-4b-DE: n = 23) using spinning-disc confocal microscopy. GFP signal was recorded every 0.2 s for 90 s. Scale bar, 20 µm. Insets show magnified lamellipodia before (pre) and after (post) photobleaching at different time points. Scale bar, 2 µm. ( B ) Relative GFP-actin fluorescence intensity changes over time. Data represent mean ± S.E.M for three independent experiments.

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b expression does not affect F-actin turnover in A375 cells. ( A ) A375 cells were transfected with GFP-actin together with one of the following plasmids, pmCherry-C1, mCherry-PMCA4b, mCherry-PMCA4b-DE, and cultured for 48 h. Media were changed to phenol free DMEM and cells were kept at 37 °C. GFP-actin was photobleached at the ruffles (mCherry: n = 8, mCherry-4b: n = 13, mCherry-4b-DE: n = 23) using spinning-disc confocal microscopy. GFP signal was recorded every 0.2 s for 90 s. Scale bar, 20 µm. Insets show magnified lamellipodia before (pre) and after (post) photobleaching at different time points. Scale bar, 2 µm. ( B ) Relative GFP-actin fluorescence intensity changes over time. Data represent mean ± S.E.M for three independent experiments.

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Cell Culture, Confocal Microscopy, Fluorescence

    PMCA4b protects F-actin from Ca 2+ -induced degradation. ( A ) Near actin Ca 2+ signal was initiated in A375 cells transiently expressing GCAMP2-actin together with mCherry-PMCA4b or mCherry-PMCA4b-DE by the addition of 2 µM A23187. GCAMP2 and mCherry signals were recorded every 15 s for 10 min using a spinning-disc confocal microscopy. Confocal microscopy images show cells before, at the peak and 7 min after the addition of A23187, as indicated. Arrowheads show GCAMP2-actin signal retraction. Scale bar, 20 µm. Graphs represent fluorescence intensity values (F/F0) of the cell shown in ( A ). Error bars represent S.E.M obtained from two independent experiments (two cells analyzed in each, two ROIs per cell). Arrows indicate the addition of A23187. ( B ) 2 µM A23187 was added to A375 cells expressing GFP-actin together with pmCherry-C1, mCherry-PMCA4b, or mCherry-PMCA4b-DE, and Z-stack images of GFP and mCherry signals were recorded every 15 s for 10 min using a spinning-disc confocal microscope. Three-dimensional confocal images were created by the ZEN 2.3 (blue edition) software, and presented at 0, 5, and 10 min. Arrowheads indicate changes in cell shape. Area and circularity parameters for A375 cells transfected with the same plasmid combinations ( n = 2–3 each) were analyzed by the ImageJ software. Data represent mean ± S.E.M. Significance is calculated for the 10-min time points (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b protects F-actin from Ca 2+ -induced degradation. ( A ) Near actin Ca 2+ signal was initiated in A375 cells transiently expressing GCAMP2-actin together with mCherry-PMCA4b or mCherry-PMCA4b-DE by the addition of 2 µM A23187. GCAMP2 and mCherry signals were recorded every 15 s for 10 min using a spinning-disc confocal microscopy. Confocal microscopy images show cells before, at the peak and 7 min after the addition of A23187, as indicated. Arrowheads show GCAMP2-actin signal retraction. Scale bar, 20 µm. Graphs represent fluorescence intensity values (F/F0) of the cell shown in ( A ). Error bars represent S.E.M obtained from two independent experiments (two cells analyzed in each, two ROIs per cell). Arrows indicate the addition of A23187. ( B ) 2 µM A23187 was added to A375 cells expressing GFP-actin together with pmCherry-C1, mCherry-PMCA4b, or mCherry-PMCA4b-DE, and Z-stack images of GFP and mCherry signals were recorded every 15 s for 10 min using a spinning-disc confocal microscope. Three-dimensional confocal images were created by the ZEN 2.3 (blue edition) software, and presented at 0, 5, and 10 min. Arrowheads indicate changes in cell shape. Area and circularity parameters for A375 cells transfected with the same plasmid combinations ( n = 2–3 each) were analyzed by the ImageJ software. Data represent mean ± S.E.M. Significance is calculated for the 10-min time points (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Confocal Microscopy, Fluorescence, Microscopy, Software, Transfection, Plasmid Preparation

    A375-GFP-PMCA4b but not the parental or the non-functional mutant expressing PMCA4b-DE cells maintain shape after Ca 2+ overload. ( A ) A375-GFP-PMCA4b cells or A375 cells transiently expressing GFP-PMCA4b-DE were treated with 2 µM A23187 in HBSS buffer containing 2 µM Ca 2+ for 10 min at 37 °C. Confocal and DIC microscopy images were taken after labeling with Phalloidin-TRITC. Scale bar, 20 µm. Right images show cells with higher magnification, scale bar, 20 µm. Arrowheads show the position of actin in relation to the cell periphery. ( B ) Area and circularity parameters were analyzed for each cell type ( n = 17–33) by ImageJ software. The mean ± S.E.M values of data are presented as a scatter plot. Confocal microscopy images of cells incubated in the presence or absence of cytochalasin D (CytD) or A23187 are shown in (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: A375-GFP-PMCA4b but not the parental or the non-functional mutant expressing PMCA4b-DE cells maintain shape after Ca 2+ overload. ( A ) A375-GFP-PMCA4b cells or A375 cells transiently expressing GFP-PMCA4b-DE were treated with 2 µM A23187 in HBSS buffer containing 2 µM Ca 2+ for 10 min at 37 °C. Confocal and DIC microscopy images were taken after labeling with Phalloidin-TRITC. Scale bar, 20 µm. Right images show cells with higher magnification, scale bar, 20 µm. Arrowheads show the position of actin in relation to the cell periphery. ( B ) Area and circularity parameters were analyzed for each cell type ( n = 17–33) by ImageJ software. The mean ± S.E.M values of data are presented as a scatter plot. Confocal microscopy images of cells incubated in the presence or absence of cytochalasin D (CytD) or A23187 are shown in (* p < 0.05, ** p < 0.01, *** p < 0.001).

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Functional Assay, Mutagenesis, Expressing, Microscopy, Labeling, Software, Confocal Microscopy, Incubation

    PMCA4b expression results in cofilin reorganization to the leading edge. ( A ) mCherry-Cofilin was transfected in A375 and A375-GFP-PMCA4b cells or cotransfected with GFP-PMCA4bDE in A375 cells. Confocal microscopy images were taken after nuclear staining with DAPI. Scale bar, 20 µm. Insets show a part of a cell with higher magnification. Arrowheads show the location of expressed cofilin at the leading edge in A375-GFP-PMCA4b cells and filopodia or protrusion in A375 cells and A375 expressing GFP-PMCA4bDE. Scale bar, 5 µm. ( B ) A375 and A375-GFP-PMCA4b cells were cultured in a 6-well plate for 48 h. Endogenous P-cofilin protein expression from total cell lysate was analyzed by Western blotting. β-tubulin was used as a loading control.

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: PMCA4b expression results in cofilin reorganization to the leading edge. ( A ) mCherry-Cofilin was transfected in A375 and A375-GFP-PMCA4b cells or cotransfected with GFP-PMCA4bDE in A375 cells. Confocal microscopy images were taken after nuclear staining with DAPI. Scale bar, 20 µm. Insets show a part of a cell with higher magnification. Arrowheads show the location of expressed cofilin at the leading edge in A375-GFP-PMCA4b cells and filopodia or protrusion in A375 cells and A375 expressing GFP-PMCA4bDE. Scale bar, 5 µm. ( B ) A375 and A375-GFP-PMCA4b cells were cultured in a 6-well plate for 48 h. Endogenous P-cofilin protein expression from total cell lysate was analyzed by Western blotting. β-tubulin was used as a loading control.

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Confocal Microscopy, Staining, Cell Culture, Western Blot

    Proper trafficking of PMCA4b is needed to maintain a front-to-rear Ca 2+ concentration gradient in A375 cells. A375, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells were transfected with the CMV-R-GECO1 plasmid. Cells were fixed and confocal microscopy images were taken. Low Ca 2+ levels are indicated by the arrowheads at the leading edge of A375-GFP-PMCA4b cells. Scale bar, 20 µm. Graphs represent GFP-PMCA4b (green) and R-GECO intensity (red) profiles for the line (red) drawn along the cells on the confocal images. Arrowheads show the GFP signal at the cell periphery. Line plots were drawn and analyzed using the ImageJ software.

    Journal: Cancers

    Article Title: The Plasma Membrane Ca 2+ Pump PMCA4b Regulates Melanoma Cell Migration through Remodeling of the Actin Cytoskeleton

    doi: 10.3390/cancers13061354

    Figure Lengend Snippet: Proper trafficking of PMCA4b is needed to maintain a front-to-rear Ca 2+ concentration gradient in A375 cells. A375, A375-GFP-PMCA4b, and A375-GFP-PMCA4b-LA cells were transfected with the CMV-R-GECO1 plasmid. Cells were fixed and confocal microscopy images were taken. Low Ca 2+ levels are indicated by the arrowheads at the leading edge of A375-GFP-PMCA4b cells. Scale bar, 20 µm. Graphs represent GFP-PMCA4b (green) and R-GECO intensity (red) profiles for the line (red) drawn along the cells on the confocal images. Arrowheads show the GFP signal at the cell periphery. Line plots were drawn and analyzed using the ImageJ software.

    Article Snippet: To generate the MCF-7-Sh-PMCA4b cell line, MCF-7 cells were transfected with the PMCA4b shRNA plasmid (sc-42602-SH, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using FuGENE HD transfection reagent (Promega, Madison, WI, USA) according to the manufacturer’s instructions.

    Techniques: Concentration Assay, Transfection, Plasmid Preparation, Confocal Microscopy, Software