Inhibition of gp130/JAK signaling fails to affect the response of SH-SY5Y cells to 6-OHDA treatment.

<p><b>A</b>, CLCF1/CRLF1 heterodimers are ligands for the ciliary neurotrophic factor receptor (CNTFR) complex, which includes the co-receptor gp130. This complex activates JAK family tyrosine kinases, prominently JAK2, which subsequently phosphorylate an array of targets including STAT family proteins STAT1 and STAT3. STAT proteins are dimeric transcription factors that translocate to the nucleus and regulate gene expression upon phosphorylation. <b>B</b>, Immunoblot analysis demonstrates that inhibition of the CNTFR/JAK/STAT signaling pathway with gp130 neutralizing antibodies (α-gp130) or the JAK1/2 specific inhibitor ruxolitinib (1 µM) blocks activation of STAT1 and STAT3 in both undifferentiated and RA/TPA differentiated SH-SY5Y cells compared to untreated control (con) cells of the same condition. Note that these inhibitors are highly specific for this pathway, and fail to impact growth/survival signaling by other key pathways including the MAPK (pERK), PI(3)K/AKT (pAKT) and mTOR (pS6) pathways. Control blots tyrosine hydroxylase (TH) are included to demonstrate differentiation, whereas blots for ERK1/2, AKT, S6 and tubulin are included to demonstrate equal protein loading. <b>C</b>, Undifferentiated SH-SY5Y cells cultured in media containing 10% FBS and α-gp130 (1 µg/mL), ruxolitinib (1 µM) or vehicle control (1 µg/mL non-specific mouse IgG or 0.1% DMSO) were concomitantly treated with different doses of 6-OHDA for 24 hours. Relative cell number was normalized to untreated cells and plotted according to 6-OHDA concentration. Dose-response curves were generated to identify the LD50 (gray dashed line) of each cellular state. LD50 values ± SE are indicated in the table below the graph along with significance scores (<i>p</i>-value) comparing antibody or drug-treated cells to controls. <b>D</b>, RA/TPA differentiated SH-SY5Y cells were treated with pathways inhibitors and 6-OHDA, and cell survival was analyzed as in C. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing antibody or drug-treated cells to controls. Note that neither inhibitor has a significant effect on the survival of cells in response to 6-OHDA.