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: Molecular Brain

Article Title: Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2

doi: 10.1186/s13041-016-0206-6

Figure Lengend Snippet: Altered synapse composition and function in RICH2 −/− mice. a Western blot analysis (showing relative percentages of mean + SEM) of hippocampal synapse-enriched P2-fractions extracted from P70 wild type (+/+, n = 3), and knock-out (−/−, n = 3) mice. Proteins were normalized to GAPDH expression levels. Right panel: Representative illustration from hippocampal P2-immunoblots. For each protein analyzed two representative immunonblot-signals are illustrated per genotype. A significant increase can be seen for GluN1 and GluN2A levels, while a significant decrease is visible for β-Actin and Cortactin (unpaired t -test, GluN1: df = 4, p = 0.0102, t = 4.4439; GluN2A: df = 4, p = 0.005, t = 5.5458; β – Actin: df = 4, p = 0.0259, t = 3.463; Cortactin: df = 4, p = 0.0264, t = 3.3999). b Western blot analysis (showing relative percentages of mean + SEM) of hippocampal S2-fractions extracted from P70 wild type (+/+, n = 3), and knock-out (−/−, n = 3) mice. Proteins were normalized to GAPDH expression levels. Right panel: Representative illustration from hippocampal S2-immunoblots. For each protein analyzed two representative immunonblot-signals are illustrated per genotype. c Western blot analysis (showing relative percentages of mean + SEM) of cerebellar synapse-enriched P2-fractions extracted from P70 wild type (+/+, n = 3), and knock-out (−/−, n = 3) mice. Proteins were normalized to GAPDH expression levels. Right panel: Representative illustration from cerebellar P2-immunoblots. For each protein analyzed two representative immunonblot-signals are illustrated per genotype. A significant increase in protein expression levels can be seen for GluN2B, mGluR5, RHOA, and CDC42 (unpaired t -test, GluN2B: df = 4, p = 0.0126, t = 4.2997; mGluR5: df = 4, p = 0.0006, t = 10.0161; RhoA: df = 4, p = 0.0011, t = 8.4287; CDC42: df = 4, p = 0.005, t = 5.5827). Note that expressions of GluN2B and mGluR5 in general were found to be very low in cerebellar lysates. d, e qRT-PCR analysis showing relative changes in hippocampal and cerebellar mRNA-levels (normalized to HMBS) using total RNA extracted from crude cellular homogenate from P70 wild type (+/+, n = 3) and knock-out (−/−, n = 3) mice. Each qRT-PCR experiment was set up of 3 biological and 3 technical replicates. The results show no significant differences of tested genes between wild type and RICH2 −/− mice in hippocampus (unpaired t -test, GluA4: df = 4, p = 0.7060, t = 0.105956; GluN1: df = 4, p = 0.1175, t = 1.9897; GluN2A: df = 4, p = 0.4582, t = 0.8201; Shank3: df = 4, p = 0.1658, t = 1.6924; Cortactin: df = 4, p = 0.6827, t = 0.4399; β actin: df = 4, p = 0.9026, t = 0.1303; RhoA: df = 4, p = 0.593, t = 0.58; CDC42: df = 4, p = 0.6591, t = 0.4756; Rac1: df = 4, p = 0.931, t = 0.0922) ( d ) and cerebellum (GluA2: df = 4, p = 0.0836, t = 2.2929; GluA3: df = 4, p = 0.7140, t = 0.3936; GluA4: df = 4, p = 0.7553, t = 0.3338; GluN2B: df = 4, p = 0.8972, t = 0.1377; mGluR5: df = 4, p = 0.8873, t = 0.1509; Shank3: df = 4, p = 0.9134, t = 0.1158; RhoA: df = 4, p = 0.4049, t = 0.9303; CDC42: df = 4, p = 0.4359, t = 0.8649; Rac1: df = 4, p = 0.9799, t = 0.0268) ( e )

Article Snippet: GTPase assays for RhoA, RAC1 and CDC42 were performed on P2 lysates of brain tissues from WT and KO animals using the RHOA / RAC1 / CDC42 G-LISA Activation Assay Bundle 3 Kits (BK 135, Cytoskeleton) according to the manufacturer's instructions and protocol.

Techniques: Western Blot, Knock-Out, Expressing, Quantitative RT-PCR

Journal: Molecular Brain

Article Title: Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2

doi: 10.1186/s13041-016-0206-6

Figure Lengend Snippet: Functional analysis of impaired synaptic signaling caused by deletion of RICH2. a Schematic illustration of small G-protein GTPase-cycle. ( G = G-protein (e.g. RHO , RAC1 , CDC42) , GAP = GTPase activating protein , GEF = guanine-nucleotide exchange factor , Pi = phosphate). G proteins are activated via GEF (guanine nucleotide exchange factor) upon replacement of GDP by GTP and inactivated via GAP (GTPase activating protein) due to hydrolysis of GTP via GAPs. b Using G-LISA activation assays, RHOA, RAC1, and CDC42 activity was measured in P2 fractions from hippocampal tissue lysates of three animals per group in technical triplicates. The results show a significantly increased activation (GTP-binding) of RAC1 and CDC42 in RICH2 −/− mice compared to wild type controls (unpaired t -test, Rac1: df = 4, p = 0.0476, t = 2.8243; Cdc42: df = 4, p = 0.0275, t = 3.3908). No significant difference was detected for RhoA (unpaired t -test, df = 4, p = 0.4727, t = 0.7919). c Western blot analysis (showing relative percentages of mean + SEM) of hippocampal synapse-enriched P2-fractions extracted from P70 wild type (+/+, n = 3), and knock-out (−/−, n = 3) mice. Proteins were normalized to GAPDH expression levels. A significant increase in LIMK1 expression levels can be observed in RICH2 −/− animals (unpaired t -test, LIMK: df = 4, p = 0.005, t = 5.6029). Given that the levels of phosphorylated LIMK1 (pLIMK1) remain unchanged, a difference in the ratio of pLIMK1 / LIMK1 can be observed, however, only as trend (pLIMK/LIMK: df = 4, p = 0.0545, t = 2.6923). In addition, the levels of EPS8 and PSD-95 are significantly increased in RICH2 −/− mice (unpaired t -test, EPS8: df = 4, p = 0.0384, t = 3.039; PSD-95: df = 4, p = 0.0529, t = 2.7223). Right panel: Representative illustration from hippocampal P2-immunoblots. For each protein analyzed two representative immunonblot-signals are illustrated per genotype. d ) Hippocampal P2 lysates from RICH2 +/+ and RICH2 −/− animals ( n = 3) were used in an actin polymerization assay. The lysate was added to a solution with pyrene-conjugated actin and the increase in fluorescence intensity that occurs when pyrene G-actin (monomer) forms pyrene F-actin measured over a time-course of 80 min. P2 lysate from RICH2 −/− mice is able to induce actin polymerization to a significantly higher amount within the first 30 min of the experiment compared to lysate from RICH2 +/+ mice (repeated measure ANOVA, effect of genotype by time F 1.14 = 5.402, p < 0.0001; effect of time F 1.14 = 6.397, p < 0.0001; effect of the genotype F 1.14 = 9.015, p = 0.040). e ) Schematic overview of RAC1 downstream signaling pathways. In RICH2 −/− mice, effects on spine morphology and actin polymerization most likely are mediated by activation of EPS8

Article Snippet: GTPase assays for RhoA, RAC1 and CDC42 were performed on P2 lysates of brain tissues from WT and KO animals using the RHOA / RAC1 / CDC42 G-LISA Activation Assay Bundle 3 Kits (BK 135, Cytoskeleton) according to the manufacturer's instructions and protocol.

Techniques: Functional Assay, Activation Assay, Activity Assay, Binding Assay, Western Blot, Knock-Out, Expressing, Polymerization Assay, Fluorescence

Journal: PLoS ONE

Article Title: The Human Minor Histocompatibility Antigen1 Is a RhoGAP

doi: 10.1371/journal.pone.0073962

Figure Lengend Snippet: (A) Sequence alignment of HMHA1 with the typical RhoGAP, p50RhoGAP, and the structurally-related BAR-GAPs, GRAF1 and OPHN1. Green indicates two matching amino acids. Pink indicates three matching amino acids. Purple indicates four matching amino acids. The arginine finger region is indicated with a black bar. (B) 3D model of the protein-protein complex between RhoA and the HMHA1 RhoGAP domain highlighting the catalytic residues (in sticks, colour coding as indicated; P-loop-Switch I-Switch II of RhoA in light green). The homology model for the GAP domain of human HMHA1 is based on the structure of the human p50RhoGAP domain (PDB ID: 1tx4), using Phyre. The position of the HMHA1 GAP domain in the complex with human RhoA (from RhoA⋅GDP⋅AlFx⋅p50RhoGAP; PDB ID: 1tx4) was obtained through its overlay on the p50RhoGAP domain. The RhoGAP domain of GRAF1 from Gallus gallus (PDB ID: 1f7c) was superimposed onto the model of the HMHA1 GAP domain. (C) HMHA1 C1-GAPtail has in vitro GAP activity towards Rac1, Cdc42, and RhoA but not towards Ras (purple bars). p50RhoGAP was used as a positive control (red bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic nucleotide hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD. (D) HMHA1 GAP activity is inhibited by the N-terminal BAR domain as full-length HMHA1 has no GAP activity while C1-GAPtail, lacking the N-terminal region, shows GAP activity (purple bars). GTPases or HMHA1 only were used as a control and as a measure for intrinsic hydrolysis (yellow bars). Data are mean values of two independent experiments. Error bars indicate SD.

Article Snippet: In vitro GAP activity of HMHA1 was measured using a RhoGAP Assay (BK105; Cytoskeleton) according to the manufacturers' recommendations.

Techniques: Sequencing, In Vitro, Activity Assay, Positive Control, Control