Journal: Journal of Cell Science

Article Title: Altering integrin engagement regulates membrane localization of K ir 2.1 channels

doi: 10.1242/jcs.225383

Figure Lengend Snippet: Effects of microtubular trafficking and endocytosis on FAKi-induced IK1 reduction in micropatterned Ex293 cells. (A) Representative images of micropatterned Ex293 cells immunostained for active β1-integrin after exposure to 12 µM Nocodazole (Noco, top) or 100 µM FAK-inhibitor (FAKi)+12 µM Nocodazole (Noco+FAKi, bottom) for 2, 4 or 6 h. ‘VC’ represents a 6-h exposure to vehicle control. (B,C) Corresponding quantifications of active β1-integrin coverage (B, n=10 cells per group; mean±s.e.m.) and IK1 density measured at −90 mV (C, n=5–14 cells per group; mean±s.e.m.). (D) Representative images of Ex293 cells stained for active β1-integrin after exposure to 25 µM Dynasore (Dyn, top) or 100 µM FAK-inhibitor (FAKi)+25 µM Dynasore (Dyn+FAKi, bottom) for 2, 4 or 6 h. (E,F) Corresponding quantifications of active β1-integrin coverage (E, n=10 cells cells per group; mean±s.e.m.) and IK1 density measured at −90 mV (F, n=6–20 cells per group; mean±s.e.m.). (G) Correlation between IK1 density and active β1-integrin coverage for different interventions. (H) Representative images of Ex293 cells stained for vinculin after exposure to 25 µM Dynasore (Dyn, top) or 100 µM FAK-inhibitor (FAKi)+25 µM Dynasore (Dyn+FAKi, bottom) for 2, 4 or 6 h. (I) Corresponding quantifications of total FA coverage in Ex293 cells (n=6–13 cells per group; mean±s.e.m.). *P<0.05, **P<0.01, ***P<0.001 for without FAKi versus with FAKi; #P<0.05, ##P<0.01, ###P<0.001 for Noco treatment compared to vehicle control and Dyn treatment compared to vehicle control; $P<0.05, $$P<0.01, $$$P<0.001 for Noco+FAKi treatment compared to vehicle control and Dyn+FAKi treatment compared to vehicle control. Note: vehicle controls are pooled for panels B–F (with and without FAKi treatment represents the same data for ‘VC’); ns, not significant (ANOVA followed by Tukey's multiple comparison test). Scale bars: 20 μm.

Article Snippet: Primary antibodies were diluted in blocking solution [0.5% (v/v) Triton-X, 1% (w/v) BSA and 3% (v/v) chicken serum] and applied overnight at 4°C and included: anti-K ir 2.1(1:100, rabbit monoclonal, ASC-026, Alomone) and anti-β1D integrin (1:200, mouse monoclonal, MAB1900, EMD Millipore) antibodies.

Techniques: Staining

Journal: Journal of Cell Science

Article Title: Altering integrin engagement regulates membrane localization of K ir 2.1 channels

doi: 10.1242/jcs.225383

Figure Lengend Snippet: Role of integrin engagement in membrane localization and function of Kir2.1 in rat cardiomyocytes. (A) Representative images of micropatterned NRVMs stained for vinculin (red), actin (green) and nuclei (DAPI, blue). Scale bar: 35 μm. (B,C) Corresponding quantifications of total FA coverage marked by vinculin (B, n=18–26 cells per group; mean±s.e.m.) and whole-cell IK1 density measured at −90 mV (C, n=7–14 cells per group; mean±s.e.m.). *P<0.05; **P<0.01; ***P<0.001 for 1600 versus 961 μm2 cells of the same shape (ANOVA followed by Tukey's multiple comparison test). (D) Representative images of micropatterned NRVMs stained for vinculin (red) and actin (green) after exposure to vehicle control for 6 h (VC) or 6 h of exposure to 10 μM Blebbistatin (Bleb, ‘B’) or 100 μM FAKi (‘F’), or plating on laminin (‘L’). Scale bar: 20 μm. (E,F) Corresponding quantifications of total FA coverage marked by vinculin (E, n=7–15 cells per group; mean±s.e.m.) and whole-cell IK1 density measured at −90 mV (F, n=6–17 cells per group; mean±s.e.m.). *P<0.05; **P<0.01; ***P<0.001 versus control (ANOVA followed by Dunnett's test). (G) Correlation between whole-cell IK1 density and total FA coverage in micropatterned NRVMs across various cell shapes, sizes and interventions. (H) Representative images of micropatterned cells immunostained for vinculin or Kir2.1–tdTomato and corresponding heat maps of average fluorescence intensity in stacked images (n=10–15 cells per group). Scale bar: 35 μm. (I,J) Corresponding quantifications of local FA coverage (I, n=10 cells per group; mean±s.e.m.) and local IK1 amplitude measured at −90 mV (J, n=3–5 cells per group; mean±s.e.m.). *P<0.05; ***P<0.001 for corner versus edge (Student's t-test). (K) Representative adult rat ventricular section immunostained for β1D integrin (red) and Kir2.1 (green) with examples of t-tubule and costameric (shown with white arrows) regions denoted with white squares. Scale bars: 20 μm. Note the lack of overlap between red and green fluorescence. (L) Magnified images of the t-tubule (left) and costamere (right) regions from K. Scale bars: 5 μm. (M) Representative green and red fluorescence intensity profiles along one of the white lines shown in L used to quantify peak-to-peak distances between Kir2.1 and β1D labels. (N) Quantification of the distance between Kir2.1 and β1D integrin puncta in t-tubule and costamere regions (n=49 fluorophore pairs per group; mean±s.e.m.). *P<0.0001 versus 0 μm distance (one-sample t-test).

Article Snippet: Primary antibodies were diluted in blocking solution [0.5% (v/v) Triton-X, 1% (w/v) BSA and 3% (v/v) chicken serum] and applied overnight at 4°C and included: anti-K ir 2.1(1:100, rabbit monoclonal, ASC-026, Alomone) and anti-β1D integrin (1:200, mouse monoclonal, MAB1900, EMD Millipore) antibodies.

Techniques: Staining, Fluorescence