Journal: Acta Biochimica et Biophysica Sinica

Article Title: LINC00891 regulated by miR-128-3p/GATA2 axis impedes lung cancer cell proliferation, invasion and EMT by inhibiting RhoA pathway

doi: 10.3724/abbs.2022005

Figure Lengend Snippet: LINC00891 regulates A549 and H460 cell functions via the RhoA pathway (A) A549 and H460 cells were transfected with LINC00891 overexpression vector, LINC00891 siRNA or respective negative controls, and the activated RhoA was determined using RhoA Activation Assay Kit. (B) Cells were treated with 15 μM CCG-1423 for 24 h, and the activated RhoA was assessed using RhoA Activation Assay Kit. Cells were treated with LINC00891 siRNA alone or together with 15 μM CCG-1423, and cell proliferation (C), invasion (D), and migration (E) were analyzed. (F) Western blot analysis was used to measure the protein levels of E-cadherin, Vimentin, Snail and Slug. n=5 in each group, *P<0.05, **P<0.01.

Article Snippet: RhoA activity was assessed using RhoA Activation Assay Kit (Merck Millipore, Billerica, USA) according to the manufacturer’s instructions.

Techniques: Transfection, Over Expression, Plasmid Preparation, Activation Assay, Migration, Western Blot

Journal: Frontiers in Molecular Biosciences

Article Title: RhoA/ROCK-YAP/TAZ Axis Regulates the Fibrotic Activity in Dexamethasone-Treated Human Trabecular Meshwork Cells

doi: 10.3389/fmolb.2021.728932

Figure Lengend Snippet: RhoAG14V promoted the expression of Collagen I and α-SMA and regulated YAP/TAZ in HTM cells (A) The bands of RhoA, α-SMA, and Collagen I proteins (B) Quantification analyses of RhoA, α-SMA, and Collagen I expression levels (C) RT-qPCR analyses of α-SMA and Collagen I mRNAs (D) The bands of YAP/TAZ, pYAP, pTAZ, and CTGF proteins (E) Quantification analyses of YAP, TAZ, pYAP, pTAZ, and CTGF proteins (F) RT-qPCR analyses of YAP, TAZ, and CTGF mRNAs (G) Representative fluorescent graphs of YAP/TAZ labeled by specific antibody and (H) quantitative analysis of YAP/TAZ in the nucleus. Scale bar, 50 μm; * p < 0.05; ** p < 0.01.

Article Snippet: The constitutively active RhoA (RhoAG14V) was provided by Beijing Syngentech Co., Ltd.

Techniques: Expressing, Quantitative RT-PCR, Labeling

Journal: bioRxiv

Article Title: Astrocytes close the critical period for visual plasticity

doi: 10.1101/2020.09.30.321497

Figure Lengend Snippet: (a) Top, volcano plot analysis representing the 2096 quantified proteins with at least 3 total peptides in all replicates enriched with biotinylated WFA lectin in KD and WT mice. Binding partners were obtained by using quantitative label-free mass spectrometry analysis performed from three replicates. Dashed vertical lines denote absolute fold change of 1.5 and the dashed horizontal line denotes the adjusted P-value of ratio significance of 0.05. Selected enriched proteins in WT (green) and KD (purple) samples are shown. Proteins from the enriched pathways Rho-GTPase activate KTN1 (blue) and Rho-GTPase activate ROCK (red) are highlighted. External plots show proteins with peptides identified only in one sample type (left in KD and right in WT). Bottom, volcano plot analysis representing interactors of Cx30 from the Rho-GTPase activate KTN1 (blue) and Rho-GTPase activate ROCK (red) pathways. The fold-change in WT (n=5) versus KD mice (n=4) are shown with selected proteins (Rock2 and Myosin 14) quantified with an absolute fold change ≥ 1.5, an adjusted P-value ≤ 0.05 and with ≥ 3 peptides. (b) Immunostaining for RhoA-GTP showed a marked increase in KD (n=27, P=0.0003), KD MD (n=16, P<0.0001) and WT MD mice (n=16, P<0.0001) compared to WT mice (n=22) (slices from 3 mice per group) (Kruskal-Wallis (KW=41.23) followed by Dunn’s post-hoc test. (c) Immunostaining for MMP9 shows a marked increase in KD mice (n=28, P<0.0001, DF=71), KD MD (n=13, P=0.0059, DF=71) and MD mice (n=12, P=0.0322, DF=71) compared to WT mice (n=22) (ANOVA (F(3,71)=15.07) followed by Tukey’s post-hoc test). (d) Diagram depicting the protocol for experimental treatment with Fasudil. (e) Fasudil rescued MMP9 levels in KD mice (KD+Fasudil: n=25 from 5 mice, KD=28 slices from 3 mice, P<0.0001), while it had no effect in WT mice (WT+Fasudil: n=34 from 6 mice, WT: 22 slices from 3 mice, P>0.9999, Kruskall-Wallis (KW=55.98) followed by Dunn’s post-hoc test). (f) Fasudil rescued perineuronal nets levels in KD mice (KD+Fasudil: n=52 from 5 mice; KD, n=177 from 16 mice, P<0.0001) while it had no effect in WT mice (WT+Fasudil, n=129 from 6 mice, WT, n=226 from 16 mice, P=0.1510) (Kruskall-Wallis (KW=71.65) followed by Dunn’s post-hoc test).

Article Snippet: The following primary antibodies were used: Cx30 rabbit polyclonal (1:500, 71-2200, Zymed), Cx43 mouse monoclonal (1:500, 610061 BD Biosciences), GFAP mouse monoclonal (1:500, G3893, Sigma-Aldrich), chick anti GFP (1:500, AB13970, Abcam), Parvalbumin mouse monoclonal (1:500, 235, SWANT), MMP9 rabbit (1:500, 3852, cell signaling), anti-active RhoA mouse monoclonal (1:500, 26904, NewEast Biosciences), Lectin from Wisteria Floribunda, biotin conjugate (1:500, L1516-2MG, Sigma-Aldrich).

Techniques: Binding Assay, Mass Spectrometry, Immunostaining