Journal: Journal of Biological Chemistry

Article Title: Expression Profiles of Arabidopsis thaliana in Mineral Deficiencies Reveal Novel Transporters Involved in Metal Homeostasis

doi: 10.1074/jbc.m309338200

Figure Lengend Snippet: FIG. 6. Functional characterization of AtOPT3. A, hybridization of an 600-bp AtOPT3 cDNA probe, corresponding to the 3-terminal exon, to RNA extracted from Arabidopsis roots of plants grown in control conditions (Ctl), copper (-Cu), manganese (-Mn), zinc (-Zn), and iron (-Fe) deficiency. The ethidium bromide-stained RNA gel is shown for quantification. B, growth of ctr1 expressing AtOPT3 on YPG-Ura plates supplemented with 10 M CuSO4 compared with the mutant transformed with the vector alone. C, growth of smf1 expressing AtOPT3 on manganese-limited medium, with and without 1 mM EGTA compared with the growth of the mutant transformed with the vector alone.

Article Snippet: Genome-wide Analysis Provides Insight into Metal Transport—We have used Affymetrix Arabidopsis DNA chips containing 8,300 genes (which cover about one-third of the ge- FIG. 7.

Techniques: Functional Assay, Hybridization, Control, Staining, Expressing, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Frontiers in Molecular Neuroscience

Article Title: Major Signaling Pathways in Migrating Neuroblasts

doi: 10.3389/neuro.02.007.2009

Figure Lengend Snippet: In vitro analysis of the cytoskeleton pathway . (A) Cytoskeleton pathway involved in neuroblast migration resulting from the microarray data analysis. (B) Boyden chamber migrational assay: dissected neuroblasts were plated on a porous membrane, were allowed to migrate for 24 hours and Tuj1-positive cells were then counted ( p < 0.001). (C,D) Migration analysis in organotypic cultures obtained from 5HT3 A -EGFP mice. (C1) PI3K inhibitor (LY294002) severely disturbed neuroblast migration. Migration was quantified as the ratio of the EGFP-positive neuroblast containing area surrounding the SVZ between untreated (control) and treated (inhibitor) slices (obtained from the same sagittal level) after 4 days in culture ( n ≥ 5 slices per condition). (C2) Higher magnifications of RMS and SVZ in control and PI3K inhibitor-treated slices. Insets: note that LY294002-treated neuroblasts have short or no neurite compared to control neuroblasts. (C3) Quantification of the LY294002 effect ( n = 5, p < 0.001). (D1) Effect of PKCζ inhibitor. (D2) Directionality of neuroblast migration and, therefore, cell polarization is disturbed after PKCζ inhibitor treatment. Neuroblasts do not migrate in the streams, but migrate in all directions. This is clearly visible in the cortex where there are significantly more neuroblasts in PKCζ inhibitor-treated slices compared to control slices. (D3) Quantification of the PKCζ inhibitor effect ( n = 6, p < 0.005) Abbreviations: Ak – Akt1 inhibitor, C – control, cx – cortex, hp – hippocampus, lv – lateral ventricle, LY – PI3K inhibitor LY294002, P3,4,5 – PIP3,4,5; P4,5 – PIP4,5; P3,4 – PIP3,4; PZ – PKCζ inhibitor, Ra – Rac1 inhibitor, TA – Rho GTPases inhibitor Toxin A of C. difficile , W – PI3K inhibitor wortmannin.

Article Snippet: The following antibodies were used in our analysis: polyclonal rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-III class β-tubulin, Tuj1, 1:500 (Covance, USA), goat anti-CaM I, 1:200 (Santa Cruz, Germany), goat anti-doublecortin, 1:500 (Santa Cruz, Germany), rabbit anti-Akt1, 1:200 (Cell Signaling, USA), mouse anti-Wave1, 1:1000 (Neuromab, USA), rabbit anti-Cdc42, 1:1000 (Santa Cruz, Germany), rabbit anti-PI3K, 1:2000 (Upstate, USA), mouse anti-Rac1 (Cytoskeleton, USA), Alexa 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, USA), anti-mouse, anti-rabbit and anti-goat Cy3 coupled secondary antibodies (Jackson Immuno Research Laboratories, USA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector, USA).

Techniques: In Vitro, Migration, Microarray

Journal: Frontiers in Molecular Neuroscience

Article Title: Major Signaling Pathways in Migrating Neuroblasts

doi: 10.3389/neuro.02.007.2009

Figure Lengend Snippet: In vivo analysis of the cytoskeleton pathway . (A) Position of injection site (arrow) and destination area of migratory fluorescent cells (oval) after 7 or 10 days post-injection. (B) Western blot analysis of transfected HEK cells illustrating successful knockdown of Wave1 , Rac1 , Pik3r1 and Akt1 . (C) Red fluorescent cells in olfactory bulb infected by shRNAScrambled and shRNAAkt1 viruses. Fewer infected cells were found in OB of shRNAAkt1-injected animals. (D) Percentage of infected cells in olfactory bulb relative to total number of infected cells on SVZ-RMS-OB route after injection of shRNA expressing viruses against genes of the cytoskeleton pathway (* p < 0.005). Gene names are under histogram. GCL – granule cell layer, GL – glomerular layer.

Article Snippet: The following antibodies were used in our analysis: polyclonal rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-III class β-tubulin, Tuj1, 1:500 (Covance, USA), goat anti-CaM I, 1:200 (Santa Cruz, Germany), goat anti-doublecortin, 1:500 (Santa Cruz, Germany), rabbit anti-Akt1, 1:200 (Cell Signaling, USA), mouse anti-Wave1, 1:1000 (Neuromab, USA), rabbit anti-Cdc42, 1:1000 (Santa Cruz, Germany), rabbit anti-PI3K, 1:2000 (Upstate, USA), mouse anti-Rac1 (Cytoskeleton, USA), Alexa 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, USA), anti-mouse, anti-rabbit and anti-goat Cy3 coupled secondary antibodies (Jackson Immuno Research Laboratories, USA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector, USA).

Techniques: In Vivo, Injection, Western Blot, Transfection, Infection, shRNA, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Major Signaling Pathways in Migrating Neuroblasts

doi: 10.3389/neuro.02.007.2009

Figure Lengend Snippet: Phenotypes of animals infected into aSVZ/pRMS by AAV viruses expressing gene-specific shRNA and red fluorescent protein .

Article Snippet: The following antibodies were used in our analysis: polyclonal rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-III class β-tubulin, Tuj1, 1:500 (Covance, USA), goat anti-CaM I, 1:200 (Santa Cruz, Germany), goat anti-doublecortin, 1:500 (Santa Cruz, Germany), rabbit anti-Akt1, 1:200 (Cell Signaling, USA), mouse anti-Wave1, 1:1000 (Neuromab, USA), rabbit anti-Cdc42, 1:1000 (Santa Cruz, Germany), rabbit anti-PI3K, 1:2000 (Upstate, USA), mouse anti-Rac1 (Cytoskeleton, USA), Alexa 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, USA), anti-mouse, anti-rabbit and anti-goat Cy3 coupled secondary antibodies (Jackson Immuno Research Laboratories, USA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector, USA).

Techniques: Infection, Expressing, shRNA

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Mixed-Lineage Leukaemia Gene Regulates Glucose-Sensitive Gene Expression and Insulin Secretion in Pancreatic Beta Cells

doi: 10.3390/ijms25094704

Figure Lengend Snippet: Decrease in insulin secretion in response to glucose loading in Kmt2a -knockdown βHC-9 cells. ( A ) Kmt2A expression was silenced via siRNA-mediated Kmt2A knockdown. Small interfering RNA targeting Kmt2A (siKmt2A) or control siRNA (Ctl) (200 nM) was introduced into βHC-9 cells via electroporation, and Kmt2A mRNA expression was quantified after 48 h via real-time polymerase chain reaction. ( B ) Glucose-stimulated insulin secretion tests were performed in Kmt2a -knockdown βHC-9 cells. ( C ) Scatter plot of the microarray data of Kmt2A -knockdown βHC-9 cells. ( D ) SLC2a1 and SLC2a2 expression. Data represent the mean ± standard error of mean from triplicate samples. Asterisks indicate significant differences from Ctl-transfected cells. The experiment was repeated twice, with similar results. The white and black columns represent the siClt- and siKmt2A-transfected groups, respectively.

Article Snippet: The TaqMan probes for Gapdh (Mm99999915_g1), Kmt2A (Mm01179235_m1), Slc2a1 (Mm05908127_s1), and Slc2a2 (Mm00446229_m1) were obtained from Applied Biosystems (Bedford, MA, USA).

Techniques: Knockdown, Expressing, Small Interfering RNA, Control, Electroporation, Real-time Polymerase Chain Reaction, Microarray, Transfection