Journal: Data in Brief

Article Title: Gene datasets associated with mouse cleft palate

doi: 10.1016/j.dib.2018.03.010

Figure Lengend Snippet: KEGG pathways enriched with a statistically significant number of genes involved in cleft palate.

Article Snippet: Regulation of actin cytoskeleton , Fgf8 Fgfr1 Itgb1 Egfr Itgb6 Pdgfc Sos1 Fgf18 Crk Itgav Fgf10 Rac1 Pdgfrb Fgf9.

Techniques:

Journal: Data in Brief

Article Title: Gene datasets associated with mouse cleft palate

doi: 10.1016/j.dib.2018.03.010

Figure Lengend Snippet: GO biological process terms enriched with a statistically significant number of genes involved in cleft palate.

Article Snippet: Regulation of actin cytoskeleton , Fgf8 Fgfr1 Itgb1 Egfr Itgb6 Pdgfc Sos1 Fgf18 Crk Itgav Fgf10 Rac1 Pdgfrb Fgf9.

Techniques: Cell Differentiation

Journal: Data in Brief

Article Title: Gene datasets associated with mouse cleft palate

doi: 10.1016/j.dib.2018.03.010

Figure Lengend Snippet: GO Molecular Function terms enriched with a statistically significant number of genes involved in cleft palate.

Article Snippet: Regulation of actin cytoskeleton , Fgf8 Fgfr1 Itgb1 Egfr Itgb6 Pdgfc Sos1 Fgf18 Crk Itgav Fgf10 Rac1 Pdgfrb Fgf9.

Techniques: Binding Assay, Protein Binding, Activity Assay, Sequencing

Journal: Data in Brief

Article Title: Gene datasets associated with mouse cleft palate

doi: 10.1016/j.dib.2018.03.010

Figure Lengend Snippet: GO cellular component terms enriched with a statistically significant number of genes involved in cleft palate.

Article Snippet: Regulation of actin cytoskeleton , Fgf8 Fgfr1 Itgb1 Egfr Itgb6 Pdgfc Sos1 Fgf18 Crk Itgav Fgf10 Rac1 Pdgfrb Fgf9.

Techniques:

Journal: Nature Communications

Article Title: The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

doi: 10.1038/s41467-021-21579-5

Figure Lengend Snippet: A Human INT 407 cells expressing a non-targeting shRNA (NT) or shRNA targeting the 3′-UTR of IQGAP1 (930-C5) were serum-starved for 4 h, then infected with C. jejuni for 45 min. Samples were collected and analyzed by immunoblot for phosphorylated ERK and total ERK in parallel. A representative immunoblot is shown. Molecular weights are indicated in kDa. B The ratio between the intensity of the phospho-ERK and total-ERK bands were quantified from three independent biological replicates and plotted. Comparing pERK/tERK in non-targeting shRNA cells with and without C. jejuni p = 0.0069, and in IQGAP1 knockdown cells p = 0.0193. C We observed that in cells expressing non-targeting shRNA there was a robust activation of Rac1 in the presence of C. jejuni , and there was not a significant amount of activation of Rac1 in the IQGAP1 knockdown cells. Each symbol represents the results from a biological replicate ( n = 3 independent samples). Comparing Rac1-GTP levels in non-targeting shRNA cells with and without C. jejuni, p = 0.0339, and in IQGAP1 knockdown cells p = 0.8751. D A similar trend was observed for the amount of Cdc42 activation, where knockdown of IQGAP1 inhibited C. jejuni -induced Cdc42 activation. Each symbol represents the results from a biological replicate ( n = 3 independent samples). Comparing Cdc42-GTP levels in non-targeting shRNA cells with and without C. jejuni, p = 0.0435, and in IQGAP1 knockdown cells p = 0.7488. E IQGAP1 knockdown cells (930-C5) were transfected with constitutive active (CA), dominant-negative (DN) or wild-type (WT) Cdc42 and Rac1, and the amount of internalized C. jejuni was measured in each condition using a gentamicin protection assay ( n = 4 independent samples). The following comparisons were made on log-transformed counts: Cdc42-CA to Cdc42-WT ( p = 0.9650), Cdc42-DN to Cdc42-WT ( p = 0.0002), Rac1-CA to Rac1-WT ( p = <0.0001), Rac1-DN to Rac1-WT ( p = 0.7861). For all charts, bars represent the mean and error bars represent the standard deviation; significant differences were determined by ANOVA followed by a two-tailed Sidak’s multiple comparisons test (* p < 0.05).

Article Snippet: For testing of Cdc42 and Rac1 activation, samples were collected and processed in accordance with the instructions provided with the Cdc42 G-LISA and Rac1 G-LISA kits (Cytoskeleton Inc., Denver, CO).

Techniques: Expressing, shRNA, Infection, Western Blot, Activation Assay, Transfection, Dominant Negative Mutation, Transformation Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

doi: 10.1038/s41467-021-21579-5

Figure Lengend Snippet: A IQGAP1 immunoprecipitations (IPs) were performed with INT 407 cells that were non-infected, infected with the C. jejuni F38011 wild-type strain, or infected with the C. jejuni ∆ ciaD mutant. Pull-downs were probed by immunoblot (IB) with antibodies for IQGAP1 and RacGAP1. The amount of RacGAP1 in the IQGAP1 complex was reduced during infection with the C. jejuni wild-type strain but not with the ∆ ciaD mutant. The amount of Erk 1/2 and Rac1 was not changed. The protein mixture used as the input for the IQGAP1 IPs were also probed for IQGAP1, RacGAP1, Erk 1/2, and Rac1 to determine initial protein abundance. Molecular weights are indicated in kDa. B Similar IQGAP1 IPs were performed with INT 407 cells that were non-infected, and infected with the C. jejuni 81–176 wild-type strain. Quantitation of the amount of RacGAP1 in three biologically independent IP experiments indicated that the amount of RacGAP1 in the IQGAP1 complex was reduced approximately 50% during infection with the C C. jejuni F38011 wild-type strain and the (* p = 0.0186) Molecular weights are indicated in kDa. D C. jejuni 81–176 wild-type strain. Bars represent the mean and error bars represent the standard deviation; significant differences from the non-infected samples were determined by ANOVA followed by a two-tailed Dunnet’s multiple comparisons test (* p = 0.0401).

Article Snippet: For testing of Cdc42 and Rac1 activation, samples were collected and processed in accordance with the instructions provided with the Cdc42 G-LISA and Rac1 G-LISA kits (Cytoskeleton Inc., Denver, CO).

Techniques: Infection, Mutagenesis, Western Blot, Quantitation Assay, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

doi: 10.1038/s41467-021-21579-5

Figure Lengend Snippet: The first step in cellular invasion is the adherence of C. jejuni to the host fibronectin via the CadF and FlpA adhesins (gray dots on the C. jejuni ). Next, the C. jejuni flagellar-dependent effector proteins, including CiaD (orange circles), are delivered to the cytosol of the host cell. The combined action of bacterial adherence and the secreted effector proteins triggers activation (phosphorylation) of the focal adhesion components FAK, Src, and paxillin, which recruits vinculin. Focal adhesion signaling is integrated into actin remodeling signaling through the proteins p130Cas, CrkII, DOCK 180, Elmo, and Rac1. Cytosolic CiaD facilitates the activation of the Erk 1/2 signaling pathway, which results in the phosphorylation of cortactin. IQGAP1 serves as a binding partner of CiaD, and it facilitates the activation of Rac1 and Cdc42, further promoting actin reorganization and bacterial uptake through the proteins WAVE2, N-WASP, and Arp2/3. CiaD promotes Rac1 activation by excluding RacGAP1 from the IQGAP1 complex, preventing the deactivation of Rac1. Other literature has also provided support for the roles of EGFR, PDGFR, PI3K, Akt, HSP90α, GPCRs, PKC, Vav2, and Tiam-1 in coordinating bacterial uptake. Purple indicates the findings from this study.

Article Snippet: For testing of Cdc42 and Rac1 activation, samples were collected and processed in accordance with the instructions provided with the Cdc42 G-LISA and Rac1 G-LISA kits (Cytoskeleton Inc., Denver, CO).

Techniques: Activation Assay, Binding Assay

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