Treatment of SH-SY5Y cells with paraquat induces JNK-dependent accumulation of TDP-43 into SGs. Cells were treated with 1 mM paraquat overnight. Where indicated, cells were co-treated with 10 μM SP600125 (JNK inhibitor), PD98059 (ERK inhibitor), SB203580 (p38 inhibitor) or D4476 (CK1 inhibitor). Cells were examined for phosphorylation of kinases by immunoblot and accumulation of TDP-43 and HuR by immunofluorescence. A: Cells were treated with paraquat and examined after overnight incubation for activation of JNK, p38 and ERK. In addition, cells were incubated with paraquat and examined at different time points from 0-480 min (8 hr) for ERK and JNK activation. Lower panels for each image indicate that total kinase expression is unchanged, upper panels indicate changes to phorphorylated forms. B: Cells were treated with paraquat in the presence or absence of kinase inhibitors and the number of TDP-43 and HuR SGs was determined. *p < 0.05, **p < 0.01. n = minimum of 500 cells counted across multiple coverslips and separate experiments for each inhibitor. C-F: Untreated, G-J: Paraquat treated, K-N: Paraquat + SP600125 showing loss of TDP-43 but not HuR SGs, O-R: Paraquat + SP600125 showing loss of TDP-43 SGs but not diffuse cytosolic TDP-43. Green = TDP-43, red = HuR, blue = DAPI. Bottom panels indicate merged images of TDP-43 and HuR panels above. Arrows indicate SGs, arrowheads indicate diffuse TDP-43. Bar = 10 μm. Representative images from two-four separate experiments performed in duplicate or triplicate.