Journal: bioRxiv
Article Title: Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons
doi: 10.1101/2023.02.17.528927
Figure Lengend Snippet: Cortical neurons were transfected with FiNGR-PSD95-GFP to tag PSD95 endogenously. Control neurons were co-transfected with mem-mCh and showed strong PSD95 puncta localisation to protrusions ( A ), which is lost when neurons were co-transfected with memNotum ( B ) or when control cultures were incubated with soluble, recombinant Notum ( C ). Puncta expression could be rescued in memNotum transfected neurons by pre-treatment with Notum inhibitor LP-922056 ( D ). ( E ) Quantification of protrusion number showed no difference between treatment groups, ( F ) PSD95-positive protrusion quantification. Neurons were stained for PSD95 and Bassoon (BSN) after transfection with either mem-mCh ( G ) or memNotum ( H ), and co-localised puncta in mCh-expressing protrusions were quantified ( K ). PSD95/BSN puncta (shown in the inset in G ) were significantly reduced in memNotum-transfected neurons compared to control, but not when control cultures were incubated with soluble, recombinant Notum ( I ). Puncta expression could be rescued in memNotum transfected neurons by pre-treatment with LP-922056 ( J ). Statistical significance was addressed using one-way ANOVA with Dunnett’s multiple comparison test to compare relevant controls within groups and Student’s t-test to compare specific combinations. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. Experiments were performed in biological triplicate, with at least 5 fields/group were analysed/replicated.
Article Snippet: Treatments with 1μg/ml of recombinant human NOTUM protein (Bio-Techne) or 100nM LP-922056 (Kind gift from ARUK DDI) were performed for 48 h prior image acquisition.
Techniques: Transfection, Incubation, Recombinant, Expressing, Staining, Comparison