rho activator i  (Cytoskeleton Inc)

Journal: iScience

Article Title: Desmosomal cadherin tension loss in pemphigus vulgaris mediated by the inhibition of active RhoA at cell-cell adhesions

doi: 10.1016/j.isci.2025.113081

Figure Lengend Snippet: Dsg3 in response to inter- and intra-cellular force changes (A) Schematic diagram of Dsg3 tension sensor (TS) and tailless control sensor (TL). TSMod was inserted into the intracellular domain of Dsg3. (B) Immunostaining images of desmoplakin (DP) for HaCaT cells expressing Dsg3 TS or TL. Cells were immunostained for DP to check for the co-expression with sensors and nuclei were labeled with DAPI. Scale bar: 10 μm. (C) Immunogold image for HaCaT cells expressing the Dsg3 tension sensor. Positive signals were observed at desmosomes, suggesting that Dsg3 tension sensors incorporated into desmosomal cadherins. Scale bar: 500 nm. (D) Fluorescent images of donor (mTFP) and acceptor (Venus) and heatmap images of FRET ratio when Dsg3 TS/Dsg3 TL expressed HaCaT cells were exposed to EGTA for 30 min, 5 mM RhoA activator (CN01) for 30 min, ROCK inhibitor (Y27632) for 12h. Scale bar: 10 μm. (E and F) Quantitative analysis of FRET ratio at the cell-cell contact shown in D. (G) Heatmap images of FRET ratio in HaCaT cells treated with the following conditions: untreated control (No Abs), 2 μg/mL AK23 (AK23-2), 10 μg/mL AK23 (AK23-10), 2 μg/mL PVIgG (PVIgG-2) and 100 μg/mL PVIgG (PVIgG-100) at 0h, 4h, 8h, and 24h. Scale bar: 10 μm. (H) Quantitative analysis of corresponding FRET ratio. All values are mean ± SD ( n ≥ 30). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ns, p > 0.05. Three repeats for all conditions.

Article Snippet: Rho activator I (CN01) (Cytoskeleton) was used at a concentration of 1 unit/mL.

Techniques: Control, Immunostaining, Expressing, Labeling

Journal: iScience

Article Title: Desmosomal cadherin tension loss in pemphigus vulgaris mediated by the inhibition of active RhoA at cell-cell adhesions

doi: 10.1016/j.isci.2025.113081

Figure Lengend Snippet: PV Ab-induced tension loss at Dsg3 inhibits RhoA (A and B) Heatmap images and quantitative analysis of RhoA FRET ratio show the activities of active RhoA in response to 2 μg/mL AK23 and 10 μM Y27632 at 4h and 24h. All values are mean ± SD ( n ≥ 30). (C) Western blot results for active RhoA and total RhoA under treatment of AK23, Y27632 and AK23+CN01 compared with controls (No Abs) for 4h and 24h. (D) Quantified western blot data for the ratio of active RhoA/total RhoA under AK23 and Y27632 treatment for 4h and 24 h, compared with controls. All values are mean ± SD ( n ≥ 3). (E–H) RhoA FRET ratio in HaCaT cells exposed to 2 μg/mL of AK23, PX4-4, PX4-3, anti-TPO, AtS13, and PVIgG at 30 min, 4h, 8h, and 24h. All values are mean ± SD ( n ≥ 30). (I) Summary of the significance of RhoA FRET ratio change over 24h for the Abs tested (blue = no change, white = significant decrease). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ns, p > 0.05. Scale bar: 10 μm. Three repeats for all conditions.

Article Snippet: Rho activator I (CN01) (Cytoskeleton) was used at a concentration of 1 unit/mL.

Techniques: Western Blot

Journal: iScience

Article Title: Desmosomal cadherin tension loss in pemphigus vulgaris mediated by the inhibition of active RhoA at cell-cell adhesions

doi: 10.1016/j.isci.2025.113081

Figure Lengend Snippet: Activation of RhoA restores the tension loss at cell adhesion induced by PV Abs HaCaT cells were with both 2 μg/mL of AK23 and 1 unit/mL CN01 at 4h and 24h, with CN01 being added in the final 30 min of the treatment. (A) CN01 increases the RhoA FRET ratio at both 4h and 24h. All values are mean ± SD ( n ≥ 30). (B) Quantified western blot data showing the ratio of active RhoA/total RhoA with AK23+CN01 treatment compared with controls (No Abs) for 4h and 24h. This quantifies the controls and AK23+CN01 western blot results shown in C. All values are mean ± SD ( n ≥ 3). (C) CN01 reduces the Dsg3 FRET ratio. All values are mean ± SD ( n ≥ 30). (D–F) Traction stress and junctional traction stress are increased in HaCaT cells treated with CN01. All values are mean ± SD ( n ≥ 30). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.005, ns, p > 0.05. Scale bar: 15 μm. Three repeats for all conditions.

Article Snippet: Rho activator I (CN01) (Cytoskeleton) was used at a concentration of 1 unit/mL.

Techniques: Activation Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Abnormal regulation of membrane-less organelles contributes to profilin1-associated ALS

doi: 10.1016/j.jbc.2025.110259

Figure Lengend Snippet: Actin filament agonist CN04 reduces neuronal toxicity caused by PFN1 deficiency . A , HEK 293 cells were transfected with the indicated siRNAs for 48 h, and then the PFN1-depleted cells were treated with 1 μg/ml CN04 for another 12 h. The cells were immunostained with an anti-Coilin (Cajal body) antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. B and C , Quantification of the Cajal body number and size per cell in ( A ). Means ± SD., ∗∗, p < 0.01, p values were determined by one-way ANOVA. D , HEK 293 cells were transfected with the siRNA against PFN1 for 48 h, and then the cells were treated with or without 1 μg/ml CN04 for another 12 h. The cells were harvested for qRT–PCR analysis. The data are presented as means ± SD from three independent experiments. ns, not significantly different, ∗∗, p < 0.01, p values were determined by one-way ANOVA. E-G , mouse primary cortical neurons were transfected with the indicated siRNAs for 48 h, then the neurons were treated with or without 1 μg/ml CN04 for another 12 h. The cells were immunostained with an anti-MAP2 antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. H , quantification of the Cajal body number per cell in ( E - G ). Means ± SD., ∗, p < 0.05, ∗∗, p < 0.01, p values were determined by one-way ANOVA. ( I - J ) Quantification of the neurite length and cell viability in more than 20 neurons ( E - G ). Means ± SD., ∗, p < 0.05, ∗∗, p < 0.01, p values were determined by one-way ANOVA. K , iPSC-derived motor neurons were transfected with the indicated siRNAs for 48 h, then the neurons were treated with or without 1 μg/ml CN04 for another 12 h. The cells were immunostained with an anti-MAP2 antibody. DAPI was used for nuclear staining. The stained cells were visualized by confocal microscopy. Scale bar, 10 μm. L and M , quantification of the number of Cajal bodies and neurite length in more than 20 neurons in ( L ). Means ± SD., ∗, p < 0.05, p values were determined by one-way ANOVA. N , the proposed working model elucidates the role of PFN1 in the context of Cajal body dynamics, Stress granule assembly, and neuronal survival.

Article Snippet: Cytochalasin D (Cyto D) was purchased from MCE (HY-N6682), MG132 was purchased from Calbiochem (474,790), CN04 was purchased from Cytoskeleton (Denver, CO), puromycin was purchased from MCE (HY-B1743).

Techniques: Transfection, Staining, Confocal Microscopy, Quantitative RT-PCR, Derivative Assay