Pseudophosphatase MK-STYX induces neurite extensions in PC12 cells.

<p>(<b>A</b>) Representative examples are presented to illustrate neurite outgrowths of PC12 cells over-expressing MK-STYX and GFP, MK-STYX_active, or pMT2 control plasmid and GFP. Cells were incubated 5 days. (<b>B</b>) Cells transfected with pEGFP, pMT2, or pMT2-FLAG-MK-STYX-FLAG plasmids were scored for neurite extensions ≥20 µm with a phase objective. Three replicate experiments were performed (n = 100 cells per experiment); the results are ± SEM. Statistical analysis was performed using ANOVA (F_2,8 = 76.10, ***p<0.0001).</p

MK-STYX induces neurites in the presence of MEK inhibitor (PD98059).

<p>Representative examples of PC12 cells over-expressing pMT2 and GFP, or MK-STYX and GFP, that were stimulated with 100 ng/ml NGF and (<b>A</b>) treated with, or (<b>B</b>) not treated with 50 µM PD98059. (<b>C</b>) Cells were scored for neurite extensions ≧20 µm. Statistical analysis was performed with t-tests for control group (pMT2 and pEGFP; **p<0.01) and experimental group (MK-STYX and pEGFP; **p<0.01).</p

Knockdown of MK-STYX prevents NGF stimulated PC12 neurite extensions.

<p>(<b>A</b>) PC12 cells were transfected with pMT2-FLAG-MK-STYX-FLAG. Transfected and non-transfected PC12 cells were lysed and immunoblots performed. Blots were probed with anti-STYXL, to detect endogenous MK-STYX, and anti-FLAG to detect over-expressed MK-STYX. (<b>B</b>) PC12 cells were transfected with shRNA against MK-STYX. Quantitative RT-PCR analysis of MK-STYX mRNA levels after knockdown with three specific shRNAs targeting unique regions of MK-STYX. MK-STYX shRNA-A provided the best knockdown of endogenous MK-STYX at 42%, compared to Clone B (MK-STYX-shRNA-B) (∼15%) or Clone C (MK-STYX-shRNA-C) (∼32%). (<b>C</b>) PC12 cells transfected with control, MK-STYX shRNA-A, or MK-STYX shRNA-C were lysed and immunoblotted. Anti-STYXL1 antibody showed that endogenous MK-STYX was down-regulated by both MK-STYX shRNA-A and MK-STYX shRNA-C relative to the scrambled negative control. The blot was stripped and probed with anti-ß tubulin as a loading control. Replicate experiments were performed. (<b>D</b>) Cells expressing negative control, MK-STYX shRNA-A, or MK-STYX shRNA-C were scored for neurite extensions ≥20 µm. Three replicate experiments were performed (n = 100 cells per experiment); error bars indicate ± SEM. Statistical analysis was performed using ANOVA (F_2,8 = 357.85, ***p<0.0001). (<b>E</b>) Representative examples of PC12 cells over-expressing shRNAs against MK-STYX (MK-STYX shRNA-A, MK-STYX shRNA-C, or scrambled negative control. The shRNA expression plasmids co-express GFP, which allows visualization of successful transfection. 24 hr post-transfection cells were stimulated with 100 ng/ml NGF, and 72 hr post-stimulation images were taken with phase contrast or fluorescence microscopy. Untransfected cells treated with NGF served as a positive control for neurite outgrowth.</p

MK-STYX decreases RhoA activation.

<p>(<b>A</b>) PC12 cells were transfected with pMT2 or pMT2-FLAG-MK-STYX-FLAG. 24 hr post-transfection cells were stimulated with 100 ng/ml NGF, and lysed at the indicated time points. Activation of RhoA was quantified by RhoA G-LISA small G-protein assay. Total RhoA was normalized by RhoA ELISA. Three replicate experiments were performed. Error bars indicate ± SEM. Statistical analysis was performed using multiple t-tests, and there was a significant increase in the percentage of active RhoA in pMT2-expressing control cells within 30 minutes (p<0.05) (<b>B</b>) PC12 cells were transfected with pMT2-FLAG-MK-STYX-FLAG, MK-STYX shRNA-A, or scrambled shRNA. 24 hr post-transfection cell were stimulated with 100 ng/ml NGF, lysed, and RhoA activation was quantified by RhoA G-LISA small G-protein assay. Total RhoA was normalized by RhoA ELISA. Three replicate experiments were performed. Error bars indicate ± SEM. Statistical analysis was performed using ANOVA (F_3.9 = 5.066, **p<0.01; *p<0.05). (<b>C</b>) PC12 cells transfected with MK-STYX, scrambled shRNA, or MK-STYX shRNA-A. 24 hr post-transfection cells were stimulated with NGF or not, and were lysed 24 hr thereafter and immunoblotted. Anti-phospho-cofilin antibody showed that MK-STYX decreased cofilin phosphorylation in non-stimulated cells relative to the MK-STYX shRNA-A. However, MK-STYX increased cofilin phosphorylation in cells stimulated with NGF relative to the MK-STYX shRNA-A. These blots were stripped and probed for cofilin as a loading control. Anti-STYXL1 antibody showed over-expressed MK-STYX relative to the scrambled control, and that endogenous MK-STYX was down-regulated by MK-STYX shRNA-A relative to the scrambled negative control. The blot was stripped and probed with anti-FLAG to detect over-expressed MK-STYX, and probed for anti-ß tubulin as a loading control. Three replicate experiments were performed.</p