Fig. 5. H₂O₂ increased cytonemes and TNT-like connections in astrocytes. (A-C) Phase contrast microscopy shows morphological changes of astrocytes. (D-G) Immunofluorescence images of Oregon Green-phalloidin-labeled F-actin astrocytes. (A,D) Untreated control; (B,E,G) treated with H₂O₂ (200 µM, 2 hours); (C,F) treated with H₂O₂ (200 µM, 2 hours) and SB203580 (20 µM, 30 minutes). (G) High magnification confocal micrograph shows the formation of TNT-like connections (arrow) and cytonemes (arrowheads). (H) The percentage of cells having TNT-like connections with other cells. The relative percentages of astrocytic connections in cultures was calculated as the ratio of the number of cells with TNT-like connections to the total number of cells in the same image. Each value is the average from at least 120 cells from three independent experiments. Treatment with H₂O₂ (200 µM, 2 hours) significantly increased the formation of TNT-like connections (P<0.01), and pre-treatment with SB203580 (20 µM, 30 minutes) significantly counteracted the formation of TNT-like connections (P<0.01). Scale bars: 20 µm in A, and 5 µm in G.