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Fig. 7. Inhibition of MAP kinase activity inhibits NF axonal transport. (A,C,D) Sequentially captured images of axonal neurites of GFP-transfected NB2a/d1 cells (A,C) or mitotracker-stained cells (D) captured at the indicated intervals (in minutes) before and after addition of the MAP kinase inhibitor PD98059 (time of addition indicated by arrow). For each axonal neurite, one of the panels captured before PD98059 addition was false-colored green and a subsequent image was false-colored red and these images were then merged (indicated by B for before); the same procedure was carried out for two images captured after addition of PD98059 (indicated by A for after). Superimposing structures appear in yellow/orange, whereas non-superimposing structures remain red or green. This provides an additional index of the relative amount of moving versus stationary structures. Note that the majority of GFP-tagged structures do not superimpose prior to addition of PD98059, but do so following its addition (A), that PD98059 did not induce a similar increase in superimposing mitotracker-labeled structures (C) and that DMSO (carrier for PD98059) did not increase the number of superimposing GFP-tagged structures. Comparisons of images in overall sequences also indicate that PD98059 stopped the movement of GFP-tagged filamentous and particulate structures (A), did not affect the movement of mitotracker-labeled structures (C), and that DMSO did not affect the movement of GFP-tagged structures (D). (B) Percentage of filamentous or particulate GFP-tagged structures exhibiting net anterograde, retrograde or lack of net movement prior to (con, control) and following a 15-minute treatment with PD98059 (PD), olomoucine (Olo) or SB202190 (SB) as indicated. Note that PD98059 and olomoucine but not SB202190 dramatically increased the percentage of non-moving GFP-tagged structures. (E, F) Percentage of GFP-tagged (E) or mitotracker-labeled (F) particles exhibiting net motion in either the anterograde or retrograde direction (mov, `moving') or no net motion (non mov, `non moving') compiled from 5-10 individual axons. Note that the carrier DMSO (Con) and PD98059 itself did not alter the translocation of mitotracker-labeled particles, whereas olomoucine stopped translocation of approximately 80% of mitotracker-labeled particles.
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