Fig 1: hUBQLN2 co-localized with TDP-43 and P62 but not with NF-κB and IκB-α. Immunofluorescence of Neuro2A cells at 48 h after transfection with (a) control plasmid, (b) pCMV-hUBQLN2WT and (c) pCMV-hUBQLN2P497H. Indicated antibodies were used according to Materials and Methods. Microscopy pictures were taken at 63× magnification. d-e Western analysis from three repeated experiments was used for quantification of (d) TDP-43 level (p = 0.0227 for hUBQLN2WT and p = 0.0032 for hUBQLN2P497H) and (e) p62 level (p = 0.0058 for hUBQLN2WT and p = 0.0130 for hUBQLN2P497H) in insoluble fraction. Results were calculated in fold change compared to proteins level in insoluble fraction of control cells. f Neuro2A cells were transfected with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H. Cells were extracted 48 h after transfection using aggregates assay protocol. The results suggested that overexpression of UBQLN2 species increased levels of UBQLN2, TDP-43 and p62 in the insoluble fraction. g Live imaging was performed on Neuro2A pFeGFP-hUBQLN2P497H and DsRED-TDP-43 co-transfected cells. Microscopy pictures were taken at 24 and 48 h after transfection. h Immunoblot of TDP-43 in cytoplasmic (C) vs nuclear (N) after transfection with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H plasmids. i HEK293 cells and Neuro2A cells were transfected with either control plasmid or pCMV-hUBQLN2P497H plasmid. Protein levels of hUBQLN2 and mUBQLN2 were measured at 48 h after transfection. Human UBQLN2 levels were similar in control HEK293 and pCMV-hUBQLN2P497H Neuro2A transfected cells. Scale bar = 25 μm
Fig 2: NF-κB activation induced by hUBQLN2 is suppressed with hUBQLN2 siRNA. Neuro2A cells were transfected with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H. Cells were treated or not with TNF-α 20 ng/ml for 4 h before luciferase assay or before collecting the cells. a Cytoplasmic (C) and nuclear (N) extraction was realized 48 h after transfection. Antibodies were use according to Materials and Methods. b Immunoblot quantification of nuclear phospho-NF-κB p65 vs p84 nuclear matrix as compared to nuclear levels in control cells (p = 0.0426, n = 3). c Neuro2A cells were stably transfected with pluc2p-NFκB-RE plasmid and then transfected with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H. Luciferase activity (NF-κB) was measured at 24 h (s < 0.0001 for hUBQLN2WT and hUBQLN2P497H, n = 6,) and (d) at 48 h (p = 0.0004 for hUBQLN2WT and p = 0.0061 for hUBQLN2P497H, n = 3). e Cells were co-transfected with control plasmid, pCMV-hUBQLN2WT or pCMV-hUBQLN2P497H and with hUBQLN2 siRNA or scrambled siRNA. Luciferase activity was measured at 48 h after transfection (p < 0.01, n = 5). f Immunoblot of UBQLN2 proteins. Upper band corresponds to mouse UBQLN2 (endogenous) and lower band to human UBQLN2 (transfected). Immunoblot was showing the reduced levels of only human UBQLN2 after co-transfection with siRNA
Fig 3: 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
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