Journal: bioRxiv

Article Title: Organizing neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels

doi: 10.1101/296731

Figure Lengend Snippet: TIRF images of a live HEK293T cell coexpressing GFP-Kv2.2 and DsRed2-ER5, prior to, and 15 min after, Latrunculin A (LatA) treatment. Scale bar in GFP-Kv2.2 Rest panel is 5 µm and holds for all panels. Graphs below show values measured from cells before and after a 15-minute treatment with 10 µM LatA. Top left graph. Mean ER-PM junction (EPJ) size per cell. Top right graph: Mean Kv2.2 cluster size per cell. Bottom left graph. Number of ER-PM junctions per cell. Bottom right graph. PCC values between Kv2.2 and DsRed2-ER5. Bars on all graphs are mean ± SD. See Figure 7-Tables 1-3 for values and statistical analyses.

Article Snippet: For experiments involving Latrunculin A (ThermoFisher Scientific, Cat# 428021100UG) treatment, Latrunculin A was diluted to 20 μM in imaging saline and added by pipette, to glass bottom dishes already containing imaging saline, to a final concentration of 10 μM.

Techniques:

Journal: bioRxiv

Article Title: Organizing neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels

doi: 10.1101/296731

Figure Lengend Snippet: TIRF image of a live HEK293T cell coexpressing GFP-Kv2.1 and DsRed2-ER5, prior to, and 15 min after, Latrunculin A (LatA) treatment. Scale bar in GFP-Kv2.1 Rest panel is 5 µm and holds for all panels. Graphs show values measured from cells before and after a 15-minute treatment with 10 µM LatA. Top left graph. Mean ER-PM junction (EPJ) size. Top right graph: Mean Kv2.1 cluster size per cell. Bottom left graph. Number of ER-PM junctions (EPJs) per cell. Bottom right graph. PCC values between Kv2.1 and DsRed2-ER5. Bars on all graphs are mean ± SD. See -Tables 1-3 for values and statistical analyses.

Article Snippet: For experiments involving Latrunculin A (ThermoFisher Scientific, Cat# 428021100UG) treatment, Latrunculin A was diluted to 20 μM in imaging saline and added by pipette, to glass bottom dishes already containing imaging saline, to a final concentration of 10 μM.

Techniques:

Journal: bioRxiv

Article Title: Cell-matrix adhesion controls Golgi organization and function by regulating Arf1 activation in anchorage dependent cells

doi: 10.1101/261842

Figure Lengend Snippet: (a) Serum-starved stable adherent (SA) WT-MEFs detached (5’ SUSP), held in suspension for 120 mins (120’ SUSP) and re-plated on fibronectin for 5 mins (5’ FN) were immuno-stained for β-tubulin (microtubule). Cross-sectional representative images for a middle (top panel) and upper plane (lower panel) for suspended and re-adherent cells are shown. A middle plane for two representative stable adherent (SA) cells is also shown. Cells stained for actin and γ-tubulin (to detect the centrosome) were deconvoluted and representative images for each time point shown in the lowermost panel. Images are representative of 30 cells imaged from 3 independent experiments. Scale bar in these images is set at 5 µm. ( b ) Stable adherent serum starved WT-MEFs, untreated (control), pre-treated with 10 µM Nocodazole or 0.5 µM Latrunculin A were pre-labeled with cholera toxin B (GM1-CTxB) detached (5’ SUSP) and held in suspension for 120 mins (120’ SUSP). Images are representative of 20 cells (Nocodazole) and 30 cells (Latrunculin A) respectively, from 2 independent experiments. Scale bar in the images is set at 5 µm. Serum-starved WT-MEFs expressing cis-medial (Man II) or trans Golgi (GalTase) marker were suspended for ( c ) 60 mins (60’ SUSP) and an additional 30 mins without (+30 ‘CNT) or with Latrunculin A (+30‘LatA) and ( d ) re-plated on fibronectin for 5 mins without (5’ FN+CNT) or with drug (5’ FN+LatA). Confocal Z stacks were de-convoluted and representative maximum intensity projections (MIP) (top panel) and zoomed (1.5X) surface rendered images are shown (bottom panel). Following deconvolution, discontinuous Golgi objects per cell for Man II and GalTase were determined using the Huygens image analysis software. The graph represents mean ± SE (17–20 cells from two independent experiments). Statistical analysis was done using the Mann Whitney’s test (p-value > 0.05). Scale bar in the images is set at 4 µm. Cells expressing cis Golgi (Man II) or trans Golgi (GalTase) marker held in suspension for ( e ) 60 mins (60’ SUSP) were held in suspension for an additional 30 mins without treatment (+30’ CNT) or treated with Nocodazole (+30’NOC) and ( f ) re-plated on fibronectin for 5 mins without drug (5’FN+CNT) or with nocodazole (5’FN+NOC). Confocal Z stack images were deconvoluted, represented as maximum intensity projections (MIP) (top panel) and surface rendered images zoomed 1.5X for clarity (bottom panel for each cell). Discontinuous Golgi objects per cell for Man II and GalTase were determined using the Huygens image analysis software. The graph represents mean ± SE (18–30 cells from three independent experiments). Statistical analysis comparing CNT and NOC were done using the Mann Whitney’s test (*** p-value <0.001). Scale bar in the images is set at 4 µm.

Article Snippet: Nocodazole (Cat. No. # M1404), Latrunculin A (Cat. No. # L5163), Brefeldin A (Cat. No. # B7651), Golgicide A (Cat. No. # G0923) were purchased from Sigma.

Techniques: Staining, Labeling, Expressing, Marker, Software