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.