Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Cx43 and Dlg1 colocalise in keratinocytes in vitro and in vivo . (A–C) Cx43 (red staining) and Dlg1 (green staining) localisation in (A) HEK293 cells, (B) HaCaT cells and (C) NIKS cells. Blue staining, DAPI-stained nuclei. White arrowheads indicate plasma membrane colocalisation of Cx43 and Dlg1. Scale bars: 10 µm. The boxed areas in the merged images are shown as enlarged images to the right-hand side. These images are representative of five separate experiments. (D) Cx43 (red) Dlg1 (green) and DAPI (blue) staining of an epidermal skin tissue section representative of tissues from three individuals. A negative control staining with rabbit IgG is shown to the left-hand side. Dotted white lines indicate the basal layer of the epithelium. White lines mark the, E, epidermis, and, D, dermis layers. White arrowheads indicate Cx43 and Dlg1 colocalisation in the dermis. A Zen Zeiss microscopy digital 2× zoom image is shown to the right-hand side. Similar images were obtained from three separate tissues. (E) Cx43 (red staining) colocalisation with Dlg1 (green staining) in adipocytes in the tissue section. Nuclei are stained with DAPI. Fat deposits in the cytoplasm of selected cells are outlined with dotted lines. All images representative of at least three repeats. Scale bars: 20 µm (main images in D, E); 10 µm (2× zoom images in D, E).

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: In Vitro, In Vivo, Staining, Negative Control, Microscopy

Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Cx43 and Dlg1 co-immunoprecipitation. (A) Western blot analysis of Cx43 and Dlg1 in HEK293, HaCaT and NIKS cells. (B) Quantification of protein levels in each cell line relative to the GAPDH loading control showing the mean±s.d. of three separate experiments. (C) Top panels, co-immunoprecipitation of endogenous Dlg1 by Dlg1 antibody H60 in each keratinocyte line. Bottom panels, co-immunoprecipitation of endogenous Cx43 using the H60 anti-Dlg1 antibody. IgG, control immunoprecipitation using an antibody of the same isotype. The heavy bands above and below the Cx43 band are antibody heavy and light chains (labelled ‘ab’). Vertical black line indicates multimerised Dlg1. (D) Western blots of input proteins: 10% of the amount of cell extract used in the co-immunoprecipitation assays. (E) Top panels, co-immunoprecipitation of endogenous Cx43 from the three cell types using an anti-Cx43 antibody C-6219 (Sigma). Bottom panels, co-immunoprecipitation of endogenous Dlg1 from the three cell types using the anti-Cx43 antibody. IgG, control immunoprecipitation using an antibody of the same isotype. An extra lane on the lower western blot for HEK293 cells shows remaining unprecipitated Dlg1 in the supernatant from the co-immunoprecipitation experiment. Vertical black lines indicate multimerised Dlg1. (F) GST pulldown experiment in HaCaT cells showing that GST–Dlg1 can interact with endogenous Cx43. All images representative of at least three repeats.

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Dlg1 depletion leads to reduced Cx43 levels and reduced appearance on the plasma membrane. (A) Graph of Dlg1 levels relative to GAPDH in HaCaT cells treated with a control siRNA or treated with an siRNA against Dlg1 (used at 40 nM). (B) Graph of Cx43 levels relative to GAPDH in HaCaT cells treated with a control siRNA or treated with an siRNA against Dlg1. Representative western blots are shown above each graph. The same GAPDH blot is shown as a loading control in each case because the data come from one western blot experiment (see Fig. S2 ). The data are the mean±s.d. from three separate experiments. (C,D) Dlg1 (green) and Cx43 (red) antibody staining of HaCaT cells treated with (C) a control siRNA (control) or (D) siRNA against Dlg1 (siDlg). In the merge panel, the brightness in the green Dlg1 channel has been enhanced to show location of residual Dlg1. The white arrow in the boxed image in D indicates Cx43 intracytoplasmic staining. All images representative of at least three repeats. Scale bars: 10 µm.

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: Western Blot, Staining

Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Dlg1 controls trafficking of Cx43 from the Golgi. (A) Graph of Dlg1 levels relative to GAPDH in HaCaT cells treated with a control siRNA or treated with an siRNA against Dlg1 (used at 20 nM). Half the concentration of siDlg1 was used in this experiment compared to the experiment in Fig. 3 to allow better visualisation of Cx43. (B) Graph of Cx43 levels relative to GAPDH in HaCaT cells treated with a control siRNA or treated with an siRNA against Dlg1. Graphs show the mean±s.d. from three separate experiments. (C) Quantification of Cx43 levels on the plasma membrane of 50 cells treated with control or Dlg1 siRNA. **** P <0.0001 (two-tailed t -test). (D,E) Cx43 (red) and calnexin (an ER-resident protein; green) antibody staining of HaCaT cells treated with (D) a control siRNA or (E) siRNA against Dlg1 (siDlg). (F) Manders’ colocalisation coefficient (MC) measurement of co-occurrence of Cx43 and ER staining. The mean is marked. *** P <0.0004 (two-tailed t -test). 50 individual cells were analysed for each treatment group. (G,H) Cx43 (red) antibody and 58K staining (green) of HaCaT cells treated with (G) control siRNA or (H) with siRNA against Dlg1 (siDlg). (I) Manders’ colocalisation coefficient (MC) measurement of co-occurrence of Cx43 and Golgi staining. The mean is marked. **** P <0.0001 (two-tailed t -test). 50 individual cells were analysed for each treatment group. In D, E, G and H, Merge+DAPI, merged imaged of red and green channels plus DAPI staining in blue to visualise nuclei. Enlarged sections of each merged image (white boxed areas) are shown on the right-hand side. All images representative of at least three repeats. Scale bars: 10 µm.

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Two Tailed Test, Staining

Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Dlg1 depletion does not alter Cx43 mRNA expression but increases Cx43 protein degradation. (A) qRT-PCR analysis of Cx43 mRNA levels in control siRNA-transfected (control) or Dlg1 siRNA-transfected (siDlg) HaCaT cells. Data are expressed as changes in Cx43 mRNA levels relative to changes in GAPDH mRNA levels (2 −ΔΔCt ). The mean±s.d. from three separate experiments are shown. ns, not significant (two-tailed t -test). (B) HaCaT cells were either mock-treated or treated with 10 mM NH 4 Cl or 10 µM MG132 for either 4 or 8 h. Cell lysates were harvested, and western blots performed to detect Dlg1, Cx43 and GAPDH, as a loading control. (C) Graph of quantification of protein levels of Cx43 in mock-transfected cells (mock), control siRNA-transfected cells (control) or in Dlg1 siRNA-transfected cells (siDlg) comparing levels in untreated (NT) and NH 4 Cl-treated cells. Data are expressed relative to GAPDH expression. Levels of Cx43 in cells not treated with NH 4 Cl are set at 1 to allow comparison of changes in Cx43 levels between control and Dlg1 siRNA-transfected cells. The fold change in Cx43 levels between NT and NH 4 Cl groups is shown to the side of the three NH 4 Cl bars. The mean±s.d. from three separate western blot experiments is shown. P <0.05 where indicated (two-tailed Mann–Whitney U-test). (D,E) Confocal immunofluorescence microscopy images of HaCaT cells treated with (D) a control siRNA or (E) with an siRNA against Dlg1 and stained with Cx43 and LAMP2 to detect the lysosomes. Cx43, red staining; Dlg1, green staining. Nuclei are stained with DAPI. Arrowheads indicate limited Cx43 and Dlg1 colocalisation. All images representative of at least three repeats. Scale bars: 10 µm (main image in D and E); 5 µm (2× zoom images in D and E). (F) Graph showing the mean±s.d. of the Manders' colocalisation coefficient quantification of Cx43 colocalisation with LAMP2; 50 cells were analysed in each of the control siRNA and Dlg1 siRNA (20 µM siRNA treatment, see Fig. 4 ) treatment groups. ** P <0.01 (two-tailed t -test).

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, MANN-WHITNEY, Immunofluorescence, Microscopy, Staining

Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Dlg1 depletion results in loss of gap junctional communication as measured by parachute assay in HaCaT cells. Calcein dye transfer between donor cells [HaCaT (A–C) or HeLa Ohio cells (D)] and acceptor HaCaT cells. (A) Mock-treated HaCaT donor cells. (B) HaCaT donor cells treated with siRNA against Dlg1. (C) HaCaT donor cells treated with carbenoxolone (CBX) to block gap junctional communication. (D) HeLa Ohio donor cells that lack Cx43 expression parachuted onto HaCaT acceptor cells. (E) Quantification of the total number of acceptor cells receiving calcein dye from a directly adjacent donor cell per total number of donor cells, normalised to values from mock-treated HaCaT donor cells. At least 140 donor cells per treatment group were analysed. Percentage reduction in dye transfer is indicated on the bars on the graph. The data are the mean±s.d. from three separate experiments. **** P <0.0001 (Kruskal–Wallis test followed by Dunn's post-hoc test).

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: Blocking Assay, Expressing

Journal: Journal of Cell Science

Article Title: The human discs large protein 1 interacts with and maintains connexin 43 at the plasma membrane in keratinocytes

doi: 10.1242/jcs.259984

Figure Lengend Snippet: Diagram of the Cx43 life cycle and consequences of Dlg1 depletion in a keratinocyte cell. Dark brown indicates the plasma membrane. The light brown circle indicates the nucleus where the GJA1/double helix portion represents the gene encoding Cx43. Cellular organelles and plasma membrane junctions are labelled. Hemichannels and gap junctions are indicated in grey. Microtubules are shown in green. Actin filaments are shown in pink. Light blue lozenges indicate the PDZ proteins ZO-1 and Dlg1. Black arrows indicate the normal Cx43 life cycle. Red arrows indicate the observed changes (up or down arrows indicate increased or decreased levels respectively) to the Cx43 life cycle upon Dlg1 depletion. Lines with a question mark indicates possible ER/Golgi-related routes to Cx43 degradation when Dlg1 levels are reduced. Created with Biorender.com.

Article Snippet: Primary antibodies used were control rabbit IgG (Sigma, Poole, UK), Cx43 C-6219 (Sigma) and anti-Dlg1 polyclonal antibody H-60 (Santa Cruz Biotechnology).

Techniques: