ATF4 deficiency alters the expression of apoptosis genes and enhances mutant SOD1 aggregation in NSC34 motoneuron cells.

<p>(<b>A</b>) NSC34 cells were stably transduced with lentiviral vectors expressing shRNA against ATF4 mRNA (shATF4) or control luciferase mRNA (shLuc). Expression of ATF4 was analyzed after treatment with 1 μg/mL tunicamycin (Tm) for indicated time. Then, expression of ATF4, CHOP, PDI and HSP90 levels (loading control) was monitored by Western blot analysis. The splicing of XBP1 mRNA is shown by RT-PCR using total cDNA from similar experiments. (<b>B</b>) shLuc and shATF4 cells were transiently transfected with expression vectors for SOD1^G85R-EGFP. After 72 h, mutant SOD1 aggregation was monitored by Western blot. Left panel: quantification of SOD1 aggregation levels normalized with HSP90. Mean and standard deviation is presented from three independent experiments. (<b>C</b>) In parallel, mutant SOD1 inclusions were visualized by fluorescent microscopy and quantified (lower panel, bar, 50 μm). A minimum of 150 cells per group was counted. Mean and standard deviation is presented from three independent experiments.