Figure 5

Simultaneous tricolor AFM-LSCM images of HEK-293 with GFP-tubulin (green), Cell-ROX (red) and Syto 82 nuclear dye (orange) showing morphological changes and altered localization of ROS after 2,4-D exposure. In the absence of 2,4-D (A–D), the tubulin network is clearly visible with a low ROS signal (red) localized to the mitochondria. The ROS signal increased in intensity after the addition of 1 mM 2,4-D (E–H) and was eventually mainly localized to the nucleus. The surface ultrastructure (D versus H) and properties (Table 1) were altered with 2,4-D exposure. Following 2 h exposure, the cells lost their protrusions, appeared rounded and more closely associated with neighbouring cells (I–L). (C and G) Are dual color images showing tubulin and ROS, and L is the triple color image showing all three signals, with convolution of orange and red signals shown in purple.