Journal: Scientific Reports
Article Title: Kv1.3 activity perturbs the homeostatic properties of astrocytes in glioma
Figure Lengend Snippet: Kv1.3 activity modulates glutamate buffering on astrocytes. ( a ) Time course of fluorescence ratio (ΔF/F0) changes induced by a puff of glutamate (1 mM for 0.5 sec, Glut puff ) onto astrocytic cultures loaded with BCECF-AM (10 μM, 45 min) and pre-treated with vehicle (n = 71) or PAP-1 (100 nM, n = 69). At peak ΔF/F0 in PAP1 = −0.08 ± 0.008 vs −0.046 ± 0.014 in Ctrl p = 0.0009, unpaired Student’s t -test). (b) Astrocytes were treated with PAP-1 (50 nM, grey circles) or not (black circles) with or without DHK (500 μM, triangles) for different times (from 2 to 45 min) and analyzed for intracellular D-[ 3 H]Asp, as described in the Methods section. Results are expressed as pCi/μg proteins and are the mean ± s.e.m. of at least 5 triplicate experiments. *p = 0.001 vs C of the correspondent time point, Holm-Sidak method One Way ANOVA. ( c ) Confocal images of astrocytes, untreated (C) or treated with PAP-1 (50 nM, 25 min), stained for plasma membrane GLT-1 (red, Hoechst in blue), scale bar 10 μm. On the right, data represent the mean fluorescence intensity of red signals per field ± s.e.m. n = 4, *p = 0.042 vs C, unpaired t -test. ( d ) Astrocytes untreated (−) or treated (+) with PAP-1 (50 nM, 25 min) were immunoprecipitated for Sumo-1 or control IgG and immmuno-blotted for GLT-1; total lysate (input) is shown. On the right, data represent the mean ± s.e.m. of optical density of sumoylated GLT-1 expressed as % of the input, *p = 0.028 vs C, unpaired t -test. ( e ) Representative time course of spontaneous Ca 2+ oscillation (F/F0) in cultured astrocytes loaded with Fluo4-AM in CTRL (left panel) and after PAP-1 application (right panel). Each trace in the panel represent a single ROI in the field. ( f ) Quantification of Ca 2+ transients before and after PAP-1 treatment. ( g ) Average ΔF/F0 of Ca 2+ transient in CTRL condition and after PAP-1 application. ( h ) Coronal brain sections of GL261-bearing mice treated with vehicle or PAP-1 (40 mg/kg/die) were stained for GFAP (green; Hoechst, in blue) and visualized at the border of the tumor (white dashed line), scale bar 20 μm. Right, data represent the mean area (in pixels) covered by GFAP + cells present at a distance up to 100 μm from the tumor border (mean ± s.e.m. n = 6, *p = 0.015, unpaired t -test).
Article Snippet: Immunofluorescence Coronal brain sections (20 μm) were washed in PBS, blocked (3% goat serum in 0.3% Triton X-100) for 1 h at RT and incubated overnight at 4 °C with specific antibodies, GFAP (1:750 –Novus Biologicals, NB300-141), Iba1 (1:750 - Wako, 019-19741), GLT1 (1:1000 – AbCam, ab41621), Kv1.3 (1:100 – Alomone Lab, AGP-005).
Techniques: Activity Assay, Fluorescence, Size-exclusion Chromatography, Staining, Immunoprecipitation, Cell Culture, Mouse Assay