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Fig. 6. Effect of Src inhibition on tau-induced alterations in actin reorganization. The top panel shows a schematic diagram of the different protocols of PP2 treatment. Micrographs show 3T3 cells transfected with tau, treated with 10 µM PP2 and PDGF, and double-labeled with anti-tau (A,C,E) and phalloidin-Alexa594 (B,D,F). Cells received either PP2 and PDGF simultaneously (A,B), PP2 45 minutes in advance of PDGF (C,D), or PP2 5 hours after PDGF addition (E,F) as described in the Results and shown in the schematic above. Bar, 10 µm. (G,H) Quantification of cells showing PDGF-induced actin fiber breakdown in the presence of PP2. Percentage of non-transfected and tau-transfected cells showing actin stress fiber breakdown in the presence of Src inhibition was determined using three protocols of PP2 treatment. PP2+PDGF indicates simultaneous addition of PP2 and PDGF with actin fiber breakdown assessed 10 minutes after addition (protocol 1). PP2
PDGF indicates 45 minutes of PP2 treatment in advance of PDGF addition, with actin fiber breakdown assessed 10 minutes after PDGF addition (protocol 2). PDGFPP2 indicates 5 hours PDGF incubation in advance of PP2 addition, with actin fiber breakdown assessed after 30 minutes PP2 treatment (protocol 3). Data for untreated cells were reproduced from Fig. 2J for comparison. (I) Effect of the tau phosphomimicking mutant on actin stress fiber breakdown. The percentage of cells transfected with the tau T231D/S235D mutant showing actin stress fiber breakdown was determined after either 10 minutes or 7 hours PDGF treatment. Significant differences are *P<0.05 and **P<0.001, respectively, between transfected and non-transfected cell percentages. Non-transfected cells are indicated with open bars, tau-transfected cells with black (G,H) or striped (I) bars.
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