Fig. 7From: BAX activation in mouse retinal ganglion cells occurs in two temporally and mechanistically distinct stepsThe 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)Back to article page