pmcherry n1  (TaKaRa)

Journal: Cell and Tissue Research

Article Title: The relationship between Connexin 43 (Cx43) and partner protein, human discs large homologue-1 (Dlg1) during wound closure in keratinocytes

doi: 10.1007/s00441-025-04030-9

Figure Lengend Snippet: Connexin 43 and Dlg1 are located in the cytoplasm of cells during wound closure. a Confocal microscopy images over time showing unwounded control cells and images taken at 0, 4, 8, 16 and 24 h post-wounding of the leading edge of a scrape wound in a confluent HaCaT cell culture. In the merged images, Cx43 is shown in magenta and Dlg1 is shown in green. Blue staining (DAPI) shows the nuclei. Dotted lines indicate the edge of the wounds. White arrowheads indicate examples of gap junction plaques in the Cx43 images. White asterisks indicate examples of Dlg1 at the plasma membrane and yellow asterisks indicate examples of cytoplasmic Dlg1 in the Dlg1 images. In the merged and enlarged merged images, white arrows indicate examples of co-location of Cx43 and Dlg1 on the plasma membrane. Yellow arrows indicate examples of intracellular co-location of Cx43 and Dlg1. Scale bar = 20 µm. b Graph of percentage co-localisation of Cx43 with Dlg1 (Manders coefficient) at 0, 8 and 16 h post-scrape wounding. c Graph of percentage co-localisation of Dlg1 with Cx43 (Manders coefficient) at 0, 8 and 16 h post-scrape wounding. * p < 0.05, ns, not significant. Calculations were carried out across five images at each time point. Each image had an average of 23 cells

Article Snippet: To generate a plasmid containing the sequence of human Cx43 with a C-terminal mCherry tag, we designed primers to clone human Cx43 into the pmCherry-N1 vector (Clontech) (primer sequences shown in Table ).

Techniques: Confocal Microscopy, Control, Cell Culture, Staining, Clinical Proteomics, Membrane

Journal: Cell and Tissue Research

Article Title: The relationship between Connexin 43 (Cx43) and partner protein, human discs large homologue-1 (Dlg1) during wound closure in keratinocytes

doi: 10.1007/s00441-025-04030-9

Figure Lengend Snippet: Changes in Cx43 and Dlg1 protein levels during scrape wound closure. a Western blots showing levels of Dlg1, Cx43 and GAPDH as a control in scrape-wounded confluent HaCaT cells. b Graph of quantification of levels of Cx43 in scrape-wounded confluent HaCaT cells. c Graph of quantification of levels of Dlg1 in scrape-wounded confluent HaCaT cells. Graphs show the actual Mean Gray Values of protein bands measured by ImageJ relative to the GAPDH loading controls. The data shown are the mean and the standard deviation from the mean of four separate experiments. ns, not statistically significant

Article Snippet: To generate a plasmid containing the sequence of human Cx43 with a C-terminal mCherry tag, we designed primers to clone human Cx43 into the pmCherry-N1 vector (Clontech) (primer sequences shown in Table ).

Techniques: Western Blot, Control, Standard Deviation

Journal: Cell and Tissue Research

Article Title: The relationship between Connexin 43 (Cx43) and partner protein, human discs large homologue-1 (Dlg1) during wound closure in keratinocytes

doi: 10.1007/s00441-025-04030-9

Figure Lengend Snippet: Cx43 and Dlg1 relocate to the cytoplasm and co-locate in cell projections in cells migrating towards the wound edge. a An image from live cell confocal immunofluorescence microscopy of HEK293 cells transfected with pmCherry-Cx43 (magenta) and pDlg1-GFP (green) at the edge of a scrape wound. Only a small number of cells co-expressed both proteins. The wound edge is indicated with a dashed line. Scale bar = 20 µm. The white box indicates cells shown in ( b – e ). b Cx43 antibody staining (magenta) in cells migrating towards the wound edge and co-expressing Cx43 and Dlg1. Arrowheads indicate Cx43 puncta in the cytoplasm. c Dlg1 staining (green) in cells migrating towards the wound edge and co-expressing Cx43 and Dlg1. d Merged image of Cx43 and Dlg1 staining in ( b and c ). The arrow indicates Cx43 localized with Dlg1 on the plasma membrane. e Phase contrast image of the area showing the Cx43 and Dlg1 co-expressing cells. Scale bars = 20 µm. f – h Images from a time-lapse live cell imaging experiment showing growth of a cell projection at the wound edge originating from a cell expressing fluorescently-tagged Cx43 and Dlg1 over a 4-h time period. Images show Cx43-mCherry (magenta) ( f ), Dlg1-GFP (green) ( g ) and merged images in HEK293 (H) cells co-expressing the proteins. The white arrow in ( f ) indicates Cx43 in the forming projection. The yellow asterisk in ( g ) indicates Dlg1 position in the cell projection as it begins to form. Yellow arrows in ( h ) indicate co-location of Cx43 and Dlg1 in the cell projection. The diameter of the projection over time is shown. Scale bars = 20 µm

Article Snippet: To generate a plasmid containing the sequence of human Cx43 with a C-terminal mCherry tag, we designed primers to clone human Cx43 into the pmCherry-N1 vector (Clontech) (primer sequences shown in Table ).

Techniques: Immunofluorescence, Microscopy, Transfection, Staining, Expressing, Clinical Proteomics, Membrane, Live Cell Imaging

Journal: Cell and Tissue Research

Article Title: The relationship between Connexin 43 (Cx43) and partner protein, human discs large homologue-1 (Dlg1) during wound closure in keratinocytes

doi: 10.1007/s00441-025-04030-9

Figure Lengend Snippet: Wound closure is delayed upon Dlg1 depletion. a Graph showing levels of Dlg1 in control and siRNA Dlg1-treated HaCaT cells. b Graph showing levels of Cx43 in control and Dlg1 siRNA-Dlg1 treated HaCaT cells. c Images from an Incucyte wound closure experiment at 0, 8 and 24 h post-scratch wounding. Control, untreated HaCaT cells. siDlg, cells transfected with 40 nM siRNA against Dlg1 24 h prior to wounding. 100 nM AnGap27, cells treated with AnGap27 30 min prior to scratch-wounding. The edges of the wounds are outlined in purple. Scale bars = 600 µm. d Graph of relative wound density (RWD) against time post-wounding in the three conditions shown in ( c ). The mean and standard deviation from the mean is shown. Six biological replicates were used for the siRNA experiments and three biological replicates for the AnGap27 experiments. e The same data are shown as in ( d ) but without the 100 nM AnGap27 data to highlight the change in relative wound density due to Dlg1 depletion

Article Snippet: To generate a plasmid containing the sequence of human Cx43 with a C-terminal mCherry tag, we designed primers to clone human Cx43 into the pmCherry-N1 vector (Clontech) (primer sequences shown in Table ).

Techniques: Control, Transfection, Standard Deviation