cdc42 glisa activation assay  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: Astrocyte targeted SMN1 gene therapy and forskolin application improves astrocyte filopodia actin defects and motor neuron synaptic dysfunction in human SMA disease pathology

doi: 10.64898/2026.03.26.714618

Figure Lengend Snippet: (A) Representative images from FiloQuant analysis used to quantify filopodia density in healthy and SMA patient astrocyte monocultures at baseline and after stimulating actin remodeling using the ATP depletion and recovery assay (NaN3 +R). CMFDA dye was used to visualize the cells (grey signal) and filopodia quantified by FiloQuant tool are highlighted in magenta. Scale bar: 30µm. (B) Imaris surface and mask tool applied on to individual healthy and SMA patient astrocyte cells at baseline and after ATP depletion and recovery assay. Scale bar: 15µm. (C) Quantification of filopodia density from FiloQuant analysis across baseline and treatment conditions for healthy and SMA patient astrocyte samples. One-way ANOVA with Bonferroni multiple comparison statistical testing; ****p<0.0001. (D) Optic density readout from CDC42-GTP G-LISA assay measuring the activated form of CDC42 across baseline and treatment conditions for healthy and SMA patient astrocyte samples. One-way ANOVA with Bonferroni multiple comparison statistical testing, p-values not statistically significant. N=5 (biological replicates), n= 4 (technical replicates).

Article Snippet: The colorimetric CDC42 G-LISA GTPase activation assay kit (Cytoskeleton, Inc) was utilized to assess the amount of active CDC42-GTP within healthy and SMA astrocyte cultures at baseline and after actin stimulation via the ATP depletion and recovery assay.

Techniques: Comparison

Journal: bioRxiv

Article Title: Astrocyte targeted SMN1 gene therapy and forskolin application improves astrocyte filopodia actin defects and motor neuron synaptic dysfunction in human SMA disease pathology

doi: 10.64898/2026.03.26.714618

Figure Lengend Snippet: (A) Schematic depicting downstream cellular mechanism activated by forskolin treatment. Forskolin application activates adenylyl cyclase (AC) at the plasma membrane, leading to an increase in intracellular levels of cyclic adenosine mono-phosphate (cAMP) and downstream activation of the protein kinase A (PKA) pathway. This can modulate SMN protein levels via phosphorylation and stabilization, in addition to actin remodeling via activation of CDC42. (B) Representative confocal images of SMN signal (grey) in SMN re-expressing SMA astrocytes at baseline (SMN:GFP UTX) and after 10µM forskolin treatment (1hr 30min incubation; SMN:GFP (F). VIMENTIN signal (magenta) was used to visualize astrocytes Scale bars: 30µm. (C) Quantification of SMN puncta in SMA patient astrocytes and SMN re-expressing SMA astrocytes and after 10µM forskolin treatment. Unpaired t-test; ns=not significant. Imaris surfaces outlining cell morphology (D) and quantification of filopodia density (E) for healthy and SMA astrocytes after forskolin treatment and forskolin treatment added during recovery period of actin remodeling (NaN3 + R(F). Imaris surfaces outlining cell morphology and quantification of filopodia density for SMN:FLAG re-expressing SMA astrocytes (F&G) and SMN:GFP re-expressing SMA astrocytes (H&I) . Conditions include baseline (UTX), forskolin treatment (F), actin remodeling via ATP depletion and recovery assay (NaN3 + R) and forskolin treatment added during recovery period of actin remodeling (NaN3 + R(F). Average filopodia density of healthy and SMA astrocyte datasets (featured in ) is depicted as threshold dotted lines in graphs. One-way ANOVA with Bonferroni multiple comparison statistical testing; **p=0.0082, ***p=0.0005. N=5 (biological replicates), n= 3 (technical replicates)

Article Snippet: The colorimetric CDC42 G-LISA GTPase activation assay kit (Cytoskeleton, Inc) was utilized to assess the amount of active CDC42-GTP within healthy and SMA astrocyte cultures at baseline and after actin stimulation via the ATP depletion and recovery assay.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics, Expressing, Incubation, Comparison