Journal: bioRxiv

Article Title: JNK signalling regulates antioxidant responses in neurons

doi: 10.1101/2020.06.05.136622

Figure Lengend Snippet: A . Total glutathione content in mature neurons treated with DEM at the indicated time and concentration. Total glutathione content is relative to ethanol treated controls and data are shown as means ± SEM (** p < 0.01; *** p < 0.001, using two-way ANOVA followed by a Dunnett’s post-hoc test; n = 3 biological replicates). B Mitochondrial toxicity was assessed using WST-1 assays in neurons treated with DEM, paraquat (PQ) and rotenone (RT) at the indicated concentrations, for 24 hours. WST-1 turnover is normalized to ethanol treated controls (Veh) and shown as means ± SEM (***p < 0.001; one-way ANOVA; n = 3 biological replicates). C H 2 O 2 production was monitored using Amplex red reagent. Neurons were treated with DEM and fluorescence measured after 24 hours. Catalase (100 nM) was added to neuronal cultures 15 min before DEM treatment. Data represents means ± SEM analysed using one-way ANOVA (*** p < 0.001, n = 3 biological replicates). Amplex red fluorescence is shown as a percentage of vehicle (0.1% ethanol) treated controls. D Prolonged oxidative stress causes dendritic loss. Representative micrographs of neurons transfected with PSD95-GFP and treated, with either 0.1 % ethanol (control) or the indicated concentrations of DEM (100μM) +/− Catalase (100nM) for 48 h. Scale bar = 50 μm.

Article Snippet: Neurons were transfected at 12 days in vitro (DIV) with PSD95-GFP (a kind gift from David Bredt [ ] and FLAG-tagged Human SRXN-1 (purchased from Origene: RC207654) for 5 h using Lipofectamine 2000 (11668019, Thermo Scientific).

Techniques: Concentration Assay, Fluorescence, Transfection

Journal: bioRxiv

Article Title: JNK signalling regulates antioxidant responses in neurons

doi: 10.1101/2020.06.05.136622

Figure Lengend Snippet: A-D Mature neurons transfected either with a PSD95-GFP expression plasmid alone (top panels) or PSD-95GFP along with plasmids expressing the catalytic (GCLC) and modifying (GCLM) subunits of Glutamate Cysteine Ligase (bottom panels). Cells were incubated with either vehicle (0.1% ethanol) or DEM (100 μM) for 48 h (scale bar = 100 μm). Insets show anti-GFP (green), anti-GCLC (red) and nuclei (blue).

Article Snippet: Neurons were transfected at 12 days in vitro (DIV) with PSD95-GFP (a kind gift from David Bredt [ ] and FLAG-tagged Human SRXN-1 (purchased from Origene: RC207654) for 5 h using Lipofectamine 2000 (11668019, Thermo Scientific).

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation

Journal: bioRxiv

Article Title: JNK signalling regulates antioxidant responses in neurons

doi: 10.1101/2020.06.05.136622

Figure Lengend Snippet: A SRXN-1 overexpression rescues DEM-induced dendritic retraction. Representative micrographs of mature neurons transfected with PSD95-GFP constructs alone (left panels) or in combination with Flag-tagged human SRXN-1 (right panels) ± 100μM DEM (48hr). Cells stained with anti-GFP (green), anti SRXN-1, (Flag antibody, red) and nuclear staining with DAPI (blue). Scale bar = 100 μm. B Overexpressed human SRXN-1 co-localises with PSD95-GFP in dendritic spines. Representative images show an overlay of GFP and Flag immunofluorescence. Magnified images of dendritic spines outlined in white boxes (PSD95-GFP+SRXN-1, scale bar = 5 μm). C Endogenous SRXN-1 protein is present at synapses. Representative western blot of synaptosome fractions prepared from mouse brain probed with antibodies for the nuclear protein FOXO3a, the pre-synaptic marker synapsin 1 (Syn1), the mitochondrial protein ATP5A1, JNK and SRXN-1 as indicated. D Schematic representation of likely signaling pathways mediating Srxn-1 transcriptional induction in response to synaptic activity and oxidative stress.

Article Snippet: Neurons were transfected at 12 days in vitro (DIV) with PSD95-GFP (a kind gift from David Bredt [ ] and FLAG-tagged Human SRXN-1 (purchased from Origene: RC207654) for 5 h using Lipofectamine 2000 (11668019, Thermo Scientific).

Techniques: Over Expression, Transfection, Construct, Staining, Immunofluorescence, Western Blot, Marker, Activity Assay