Fig. 6

Characteristics of BAX translocation and permeabilization in cells after direct inhibition of Focal Adhesion Kinase (FAK) with PF573228. A Quantification of the percentage of GFP-BAX cells that translocate BAX 1 day after intravitreal injection of PF573228. Data are compared to percentages of cells induced by optic nerve crush (ONC) shown in Fig. 1 (grey bars). PF573228 induces an increase in the formation of punctate cells that is significantly greater than DMSO vehicle injections alone (***P < 0.001, ANOVA), but the level is irrespective of the dose of PF573228 injected (n.s., not significant). PF573228 induces a similar level of translocation observed 1 day after ONC (#P = 0.6404, ANOVA), but less than observed 5 days after ONC (**P = 0.0031, ANOVA). B Translocated BAX induced by PF573228 is predominantly in the paused state (6A7 staining negative) 1 day after injection, at levels significantly exceeding those observed 1 day after ONC (**P = 0.0004, *** P < 0.0001, individual t-tests). DMSO treated retinas (not graphed) yielded a total of 14 punctate cells in 7 retinas. Of these, 8 cells (57%) stained with the 6A7 antibody. C,D Retinal whole mounts 1 day after ONC (C) or PF573228 injection (D) stained for pJUN (red). Both treatments induce pJUN accumulation in cells of the ganglion cell layer, although they are more abundant in ONC. Scale bar = 60 µm. DAPI counterstain. (E, F) High magnification of two RGCs with punctate GFP-BAX (green) and exhibiting robust pJUN accumulation (E) or absence of pJUN accumulation (F). Scale bar = 7 µm. DAPI counterstain. G Histogram showing the percentage of total neurons (mean ± SD) expressing pJUN 1 d after ONC compared to 1 d after DMSO or PF573228 injection (**P = 0.0006, *** P < 0.0001, individual t-tests). H Histogram showing the distribution of cells with punctate GFP-BAX that exhibit pJUN accumulation in both ONC and PF573228 retinas (mean ± SD). Significantly more cells in ONC retinas with BAX translocation were also pJUN positive (**P = 0.0007, t-test). I Scatter plots of maximal axial length of GFP-BAX expressing cells. Measurements were taken of diffusely labeled cells for control retinas and punctate labeled cells for ONC and PF573228 exposed retinas. Both ONC and PF573228 induce BAX translocation in larger cells relative to the population of transduced cells with diffuse BAX in control retinas, 1 day after ONC or injection (***P < 0.0003, ANOVA). Due to the lack of punctate cells induced by DMSO injection, these data were not graphed. There was no significant difference (n.s., P = 0.119, t-test) between cell sizes in the two experimental groups. Cohorts contained between 48–361 cells measured from retinas of minimum of 3 mice per group. J Confocal Z-stack of a cell with partial GFP-BAX translocation in its dendritic arbor induced by PF573228 injection (Z plane shown in lower panel). An asterisk indicates the cell soma. Translocation is observed in regions of the arbor but is not fully extended to the soma, other primary branches and some secondary branches from a branch that is punctate. Axons from different labeled RGCs are indicated. Scale bars = 50 µm