Fig. 7
The effect of kinase inhibitors on the processes of BAX translocation and activation. Two inhibitors that are predicted to affect elements of the DLK signaling axis (SB2035580 to inhibit p38/MAPK14 and Sunitinib as a broad spectrum inhibitor) were injected into eyes immediately after optic nerve crush (ONC) surgery. A Immunostaining for nuclear accumulation of pJUN, 1 day after ONC surgery, shows both inhibitors have no effect on this surrogate for JUN activation. Scale bar = 15 µm. B, C Quantitative PCR analysis of pJUN (B) and p53 (C) target genes 5 days after ONC. Similar to pJUN staining results, neither of the inhibitors prevented normal increases in transcript abundance from both transcription factors. Each point represents the mean of 3 technical replicates of a sample of 4 pooled retinas. The # indicates experimental groups where one of the replicates was omitted due to nothing being detected in the qPCR run. D Quantification of the percentage of transduced cells exhibiting GFP-BAX translocation at 5 days after ONC. Neither inhibitor suppressed the translocation response, which were significantly increased relative to contralateral eyes (Con) (***P < 0.0001 for each ONC group in individual comparisons by t-test). E Analysis of BAX activation by 6A7-immunostaining showed that both inhibitors suppressed permeabilization of translocated BAX relative to ONC alone or ONC with DMSO vehicle injection (ANOVA, *** P < 0.0001). Notably, Sunitinib significantly reduced BAX permeabilization relative to SB203580 (t test, ** P = 0.0053) even though it is not reported as a p38/MAPK14 inhibitor [74]. F Quantification of the percentage of transduced cells exhibiting GFP-BAX translocation 5 days after ONC, comparing Mkk4fl;Mkk7fl Tg mice without prior introduction of Cre recombinase by AAV2-mediated gene transduction (considered wild type, WT) with Mkk4fl;Mkk7.fl mice exposed to AAV2-Cre prior to crush surgery (Mkk4/7 dKO). WT mice exhibit an ONC-induced increase in cells showing translocation relative to contralateral eyes (***P < 0.0001). Mice conditionally lacking function MKK4 and MKK7 exhibit significantly fewer punctate cells after ONC, relative to WT animals (***P < 0.0001) and no discernable change in translocating cells relative to contralateral eyes (n.s., not significant)