CRLF1 is required for differentiation-induced resistance to 6-OHDA independent of the CNTF receptor.

<p>A, Relative expression of CRLF1 was determined by quantitative RT-PCR in in stably selected SH-SH5Y cell lines containing control (non-targeting shRNA, NT-sh) and CRLF1-targeted shRNAs. Expression was normalized relative to basal expression in undifferentiated control cells. Error bars indicate standard deviation in replicate (n = 3) samples. <b>B</b>, Survival of the indicated stable shRNA cell lines in response to increasing doses of 6-OHDA. 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 CRLF1-shRNA expressing lines to NT-sh. <b>C</b>, Different concentrations of 6-OHDA diluted in conditioned media harvested from RA/TPA differentiated cells of each of the indicated stable shRNA lines were used to treated naïve SH-SY5Y cells. Dose-response curves were generated to identify the LD50 (gray dashed line) of cells treated with each media condition. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing CRLF1-shRNA expressing lines to NT-sh. <b>D</b>, Immunoblot analysis of SH-SY5Y cells treated for 15 minutes with 5 ng/mL CLC/CLF ± the indicated pre-treatment (1 hour) dose of 6-OHDA indicates that STAT3 is phosphorylated in response to the ligand independent of 6-OHDA concentration. Total STAT3 and tubulin were used as controls to demonstrate equal protein loading. <b>E</b>, Survival of the indicated stable shRNA cell lines in response to increasing doses of 6-OHDA in media ±10 ng/mL recombinant CLC/CLF ligand. LD50 values ± SE are indicated in the table below the graph along with <i>p</i>-values comparing CLC/CLF-treated cells to untreated NT-sh cells. <b>F</b>, The indicated SH-SY5Y stable shRNA lines were plated at equal densities and differentiated over six days in NBA media ±10 ng/mL recombinant CLC/CLF. Error bars indicate standard deviation in replicate (n = 6) samples.