Fig. 5
From: Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells
UBQLN2 induced a NF-κB-mediated neuronal cell death and an ER-stress response. a Neuro2A cells were transfected with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H and collected after 48 h including 4 h TNF-α 20 ng/ml treatment, (C) cytoplasmic and (N) nuclear. Quantification of (b) cleaved-caspase-3 (n = 3) and (c) caspase-12 (n = 3) vs GAPDH as compared to cytoplasmic level in control cells when treated with TNF-α. (d) Total proteins extract was used for western analysis of ER-stress marker 48 h after transfection of control plasmid pCMV-UBQLN2WT and pCMV-UBQLN2P497H. (e) Neuro2A cells were stably transfected with pluc2p-NFκB-RE plasmid and then treated with Withaferin A (WA) 0.5 μM for 2 h previous to TNF-α 20 ng/ml treatment. Luciferase activity (NF-κB) showed a significant decreased in NF-κB activity after WA treatment (n = 3, p < 0.0001). (f-h) Immunofluorescence of Neuro2A-transfected cells fixed in PFA 4 % at 48 h and then labeled with cleaved caspase-3 (green) and flag (UBQLN2) (red) in (e) control transfected cells (f) hUBQLN2WT-transfected cells and (g) hUBQLN2P497H-transfected cells. Scale bar = 25 μm. (i) MTS assay was realized on cells at 48 h after transfection. The number of live cells was represented using percentage of live cell as compared to control transfected cells. We observed significant mortality in hUBQLN2WT (p = 0.0144, n = 3) and hUBQLN2P497H (p = 0.0067, n = 3) transfected cells. Cells were also treated with Withaferin A 0.5 μM for 2 h previous to TNF-α 20 ng/ml treatment