Fig 2: AACOCF3 treatment does not decrease cPLA2 pathway protein content. (a) Representative Western blot images and (b) quantifications in whole muscle homogenate and mitochondrial fraction of sham and denervated gastrocnemius muscles from male mice treated with 7 days of corn oil or AACOCF3 injections (n = 5–6). Ponceau images of each blot were taken, then the blots were cut to probe with individual primary antibodies. n = 2 samples were run per blot. Multiple blots were compared by loading a control sample on each blot and normalizing the probed bands to the loading control and then to the control sample. The blots were processed in parallel. Full blot images are available in Supplemental Fig. 4. Statistical significance determined by two-way ANOVA with Tukey’s post hoc test for each protein. All plots represent mean ± standard deviation. *p < 0.05 for designated comparison.

Fig 3: Macrophage cytokine profiles indicate a mixed neuroinflammatory phenotype. Supernatants from treated BMDMs were collected to measure pro- and anti-inflammatory cytokine production in response to phenotype, myelin stimulation, and cPLA2 inhibition with PACOCF3. (A, B) Myelin significanty increases the production of TNF-alpha in M1 cells, but has no effect on CTL and M2 cells. cPLA2 inhibition significantly reduced myelin potentiation of M1 TNF-alpha production. (C, D) Myelin increases CX3CL1 in CTL, M1, and M2 cells. cPLA2 inhibition significantly reduced this effect in M1 cells, but not under M1 and Myelin co-stimulation. (E, F) Myelin significantly decreased IL-10 production in M1 cells, but this effect was not reversed with cPLA2 inhibition. cPLA2 inhibition alone increased IL-10 production in M1 cells. (G, H) Myelin significantly reduced pro-inflammatory IL-1beta production in M1 cells, whereas CTL and M2 cells were unaffected. cPLA2 inhibition did not alter this effect. (I, J) Pro-inflammatory IL-6 was not significantly altered by myelin or cPLA2 inhibition. Refer to Supplementary Fig. 5 for CTL and M2 cPLA2 inhibition data. Representative of three biological replications. *p < 0.05 **p < 0.01, ***p < 0.001 mean ± SEM.

Fig 4: Myelin increases cPLA2 activity in an activation state dependent manner. Cell lysates were collected from BMDM cultures to measure cPLA2 activity following stimulation into CTL, M1, and M2 cells with or without myelin. Samples containing Bromoenol Lactone, an inhibitor of iPLA2 were used to exclude the possibility of iPLA2 contributing to enzymatic activity. (A) CTL treated cells had no discernable activity with or without myelin and Bromoenol Lactone. (B) M1 treated cells had a mean enzymatic activity of 4.88 nmol/min/mL. Addition of myelin significantly increased cPLA2 enzymatic activity (*p < 0.05). Addition of Bromoenol Lactone did not significantly alter enzyme activity. (C) M2 treated cells had no discernable activity with or without myelin and Bromoenol Lactone. Representative of three biological replications. *p < 0.05 **p < 0.01, ***p < 0.001 mean ± SEM.

Fig 5: Pemetrexed-instigated release of AA activated NFkB. A. Protein levels of IKB detected by ELISA in MPM cells, pre-treated or not with a cPLA2 inhibitor (AVX001, 3 micromol/L for 90 min), and treated as indicated. Statistics: * p < 0.05 ** p < 0.01. ns = not significant (p > 0.05). B. Representative box plots showing the nuclear content of the NFkB subunit p65 bound to a synthetic NFkB responsive element and detected by ELISA in cell extracts of HP1 (left panel), NCI-H2373 (middle panel) and MSTO-211H (right panel). Statistics: * p < 0.05; ns = not significant (p > 0.05)