dominant negative rab7  (Addgene inc)

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A and B ) Affibody-chase experiments. Cells surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP) to stimulate α V β 6 integrin and trigger α V β 6 endocytosis, or vehicle (Control), 0- to 60-min time course. Quantitation represents cytoplasmic HER2 fluorescence intensity analysis in (A) trastuzumab-sensitive or (B) trastuzumab-resistant BT474 cells ( N = 3; 27 to 50 cells per condition), normalized to control trastuzumab-sensitive BT474 cells (0 min); scale bar, 10 μm. Two-way ANOVA with Šídák’s multiple comparison test. Image intensity increased in (B), relative to (A), due to low cell surface HER2 levels in trastuzumab-resistant cells to highlight internalization differences. ( C ) HER2 (green) and RAB5 (magenta) immunofluorescence in trastuzumab-sensitive and trastuzumab-resistant BT474 cells, treated with soluble LAP, 0 to 60 min ( N = 3; 16 to 28 cells per condition); scale bar, 10 μm. ( Ca ) HER2/RAB5 colocalization quantitation (Pearson’s coefficient ± SEM). Two-way ANOVA with Dunnett’s multiple comparison test. ( D ) Active RAB5 pull-down assays. 0- to 60-min LAP stimulation time course. Quantitation of mean RAB5 activity (pull-down eluate), relative to total RAB5 (lysate) ± SEM ( N = 3), normalized to 0-min trastuzumab-sensitive cells. One-way ANOVA with Dunnett’s multiple comparison test. ( E and F ) Affibody-chase experiments in (E) siControl Trastuzumab-Sensitive or (F) Trastuzumab-Resistant BT474 cells expressing constitutively active RAB5 (RAB5CA), dominant-negative RAB5 (RAB5DN), dominant-negative RAB7 (RAB7DN), or mCherry vector control. Cells were surface labeled with FITC-conjugated HER2 affibody and stimulated with soluble LAP (LAP), or vehicle control (control), for 0 or 30 min. Quantitation represents cytoplasmic HER2 fluorescence intensity ( N = 3; 81 to 87 cells per condition); scale bar, 10 μm. One-way ANOVA with Tukey’s multiple comparison test. Representative images in fig. S10 (A and B). Further HER2 internalization analyses: Supplementary Results and fig. S11 (A to D). [(A), (B), and (D) to (F)] Data are arbitrary units (AU) normalized to control means ± SEM. [(A) to (F)] Statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Labeling, Control, Quantitation Assay, Fluorescence, Comparison, Immunofluorescence, Activity Assay, Expressing, Dominant Negative Mutation, Plasmid Preparation

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Trastuzumab-Sensitive Cells: GDI2 is recruited to sites proximal to α V β 6 IACs and coordinates HER2 and α V β 6 trafficking and signaling by locally modulating RAB5 activity. GDI2-mediated cross-talk between α V β 6 and HER2 affects membrane availability of both receptors, ultimately influencing migration, invasion, and TGFβ activation. ( B ) Trastuzumab-Resistant Cells: GDI2 is excluded from α V β 6 IACs, leading to dysregulation of RAB5 activation dynamics, followed by increased RAB7 activation. Consequently, HER2/α V β 6 cross-talk is impaired, altering receptor trafficking dynamics and disrupting bioavailability of both HER2 and α V β 6 integrin at the plasma membrane. This dysregulation further affects TGFβ activation, resulting in increased cell invasiveness and metastatic potential. Overall, these changes may increase the ability of cells to evade HER2 targeting drugs.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Activity Assay, Membrane, Migration, Activation Assay, Clinical Proteomics

Journal: Science Advances

Article Title: A trafficking regulatory subnetwork governs α V β 6 integrin-HER2 cross-talk to control breast cancer invasion and drug resistance

doi: 10.1126/sciadv.adk9944

Figure Lengend Snippet: ( A ) Differential gene expression data (RNA-seq) for the GDI2 / RAB5A / RAB7A / ERBB2 / ITGB6 cluster in normal breast tissue ( n = 403; light gray) and breast invasive carcinoma ( n = 1097; dark gray). Data were extracted from the TNMplot database ( tnmplot.com ). Black lines in violin blots represent the median. Mann-Whitney test. ( B ) Volcano plot showing statistical analysis (ANOVA) of RNA-seq gene expression data of patients with HER2+ breast cancer from the METABRIC cohort expressing high (Right) and low (Left) levels of ITGB6 (Q1 versus Q4). Significant genes (dark gray); nonsignificant genes (light gray); relevant genes are highlighted in purple. ( C ) Visual representation of GO terms analysis (ClueGO, cellular compartment) of genes highly and significantly expressed in tumors expressing high levels of ITGB6 (Q4). Colors represent specific merged GO term groups, node size represents the level of significance of each GO term, and clustering and edge length represent functionally grouped networks based on kappa score. ( D ) OS of patients with HER2+ breast cancer and with high (above median) expression of ITGB6 , expressing high (red) or low (black) levels of GDI2 , ERBB2 , RAB5A , and RAB7A . ( E and F ) Differential ITGB6 gene expression (gene chip) in patients with HER2+ breast cancer subdivided according to therapeutic response to trastuzumab. (E) Initial pathological complete response (responder) versus residual disease after completing therapy (nonresponder) ( n = 77 patients). (F) RFS at 5 years (responder) versus samples relapsed before 5 years (nonresponder) ( n = 24 patients). Two-sided Student’s t test. [(A), (E), and (F)] Statistical significance: * P < 0.05; **** P < 0.0001.

Article Snippet: For protein expression, cells were transfected with DNA (1 μg/ml): constitutively active RAB5 ( ) [mcherry-RAB5CA(Q79L), Addgene plasmid #35138], dominant-negative RAB5 [mCherry-RAB5DN(S34N), Addgene plasmid #35139] , dominant-negative RAB7 [DsRed-RAB7 DN(T22N), Addgene plasmid #12662] , or empty pmCherry-C1 vector (Clontech, Addgene plasmid #3552).

Techniques: Gene Expression, RNA Sequencing, MANN-WHITNEY, Expressing, Clinical Proteomics

Journal: Science Advances

Article Title: Endosomes serve as signaling platforms for RIG-I ubiquitination and activation

doi: 10.1126/sciadv.adq0660

Figure Lengend Snippet: ( A ) HeLa cells transfected with GFP–RIG-I and TAPE-RFP were mock-treated or transfected with 5′-triphosphate RNA (5′ppp RNA; 1 μg/ml) for 4 hours. These cells were subjected to confocal microscopic analyses. Arrows indicated the merged speckles. ( B ) Similar to (A), FITC-labeled polyI:C (1 μg/ml) was transfected into HeLa cells expressing RIG-I–CFP and TAPE-RFP for 1.5 hours. The outlined area is enlarged to show merged speckles (right). ( C ) Confocal images of HeLa cells transfected with GFP–RIG-I and Rab5-RFP before and after polyI:C transfection for the indicated times (left). The outlined regions are magnified (middle). The fluorescent intensity of the white lines was measured in the areas (right). ( D ) Confocal images of HeLa cells transfected with GFP–RIG-I and Rab7-RFP before and after polyI:C transfection for the indicated times (left). The outlined regions are magnified (right). ( E ) Confocal images of HeLa cells transfected with GFP–RIG-I and FLAG-MAVS before and after polyI:C transfection for the indicated times (left). FLAG-MAVS was immunostained by the anti-FLAG antibody. ( F ) HEK293T cells were transfected with Flag–RIG-I alone or with RFP-Rab5 and then left untreated or treated with IAV RNA transfection for 3 hours. The IP-WB analysis examined RIG-I interaction with Rab5. ( G ) Confocal images of HeLa cells transfected with TAPE-CFP, GFP–RIG-I, and RFP-Rab5 before and after 5′ppp RNA (1 μg/ml) stimulation. Arrows denote speckles formed by TAPE-CFP, GFP–RIG-I, and RFP-Rab5. Scale bars, (A to E and G) 10 μm.

Article Snippet: Zhang; and DsRed-rab5 DN (Addgene, plasmid #13051) and DsRed-rab7 DN (Addgene, plasmid #12662) by R. Pagano.

Techniques: Transfection, Labeling, Expressing

Journal: The Journal of Clinical Investigation

Article Title: NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis

doi: 10.1172/JCI136042

Figure Lengend Snippet: (A) Gene expression for the late endosome marker RAB7A in CD8+ Tregs (RT-PCR, n = 10). (B) Quantification of CD8+ Treg–produced exosomes (n = 10 controls, 7 patients). (C) NOX2 protein expressed on secreted exosomes. Representative histograms and FACS results from 8 samples each. (D–F) GCA CD8+ Tregs were transfected with NOTCH4 or control siRNA. (D) RAB7A transcripts. RT-PCR from 6 patients. (E) Secreted exosomes from n = 7 samples. (F) Exosomal NOX2 protein. Flow cytometry from 6 samples. (G–I) Healthy CD8+ Tregs were transfected with an N4ICD plasmid or empty vector. (G) RAB7A transcripts quantified by RT-PCR in 6 samples. (H) Secreted exosomes determined in 8 samples. (I) Exosomal NOX2 protein. Flow cytometry from 6 samples. (J and K) Control CD8+ Tregs (J) and GCA CD8+ Tregs (K) were transfected as indicated. (J) HES5-containing or empty vector; (K) HES5 or control siRNA. RT-PCR for RAB7A transcripts in n = 6 samples. (L) ChIP assays targeting HES5 or control IgG were performed on the promoter of RAB7A or a negative site. Signal normalized to 10% of input. Data from 6 GCA CD8+ Treg samples. (M) GCA CD8+ Tregs were transfected with HES5 or control siRNA. Occupancy of HES5 on the RAB7A promoter was examined by ChIP assay. Signal normalized to 10% of input. n = 6 samples. (N) GCA CD8+ Tregs were treated with the lysosomal inhibitors chloroquine (CQ) and leupeptin (Leu), the proteasome inhibitor MG132, or vehicle. Cell surface NOX2 was evaluated by FACS. Representative histograms and results from 5 samples. (O) GCA CD8+ Tregs were transfected with a RAB5DN-containing, a RAB7DN-containing, or control vector. Lysosome intensity was measured by LysoTracker. Representative histograms and FACS results from 6 samples. CD8+ Treg cells were induced ex vivo. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired (A–C) and paired (D–K and M) Mann-Whitney-Wilcoxon rank test, or ANOVA and post-ANOVA pairwise 2-group comparisons conducted with Tukey’s method (N and O).

Article Snippet: RAB5DN (13051) and RAB7DN (12662) plasmids were obtained from Addgene. siRNAs targeting human NOTCH4, HES5, and RAB5A were purchased from Santa Cruz Biotechnology, Inc.

Techniques: Gene Expression, Marker, Reverse Transcription Polymerase Chain Reaction, Produced, Transfection, Control, Flow Cytometry, Plasmid Preparation, Ex Vivo, MANN-WHITNEY

Journal: Nature Communications

Article Title: TGFβ signalling acts as a molecular brake of myoblast fusion

doi: 10.1038/s41467-020-20290-1

Figure Lengend Snippet: a , b , d , e DML-derived myocytes electroporated with MLC promoter driving expression of dominant negative (DN) variants of RAB11 ( b ) and RAB7 ( e ), together with membrane GFP (green) and nuclear mCherry (red). c , f , Column graph for a , b and d , e showing the population of electroporated myocytes containing the indicated number of nuclei relative to their controls (in %). g – i Functional rescue experiment where DML-derived myocytes were co-electroporated with a RFP-tagged form of TGFBR2 and an inducible (Tet-on) HA-tagged form of a constitutively active RAB7. g immunostaining against RFP showing the punctated expression of TGFBR2. h immunostaining against HA showing the diffuse expression of CA RAB7 (after Doxycyclin treatment). i Native fluorescence of H2B-BFP fusion protein, showing the nuclei within electroporated myocytes. j Merge of Fig. 5g–i. k Column graph showing the population of electroporated myocytes containing the indicated number of nuclei relative to their controls (in %) in each of the indicated conditions. Statistical analyses: DN RAB11: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.57; n = 14; Ctrl: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.07; n = 15; P -value <0.0001; DN RAB7: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 1.96; n = 19; Ctrl: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.46; n = 35; P -value < 0.0001; TGFBR2: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 1.63; n = 15; Ctrl: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 2.05; n = 23; P -value <0.0001; TGFBR2 + CA RAB7: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\bar x$$\end{document} x ¯ : 1.84; n = 27; P -value=0.0019. *** P < 0.001. ** P < 0.01. Error bars in c , f , k : SEM. Scale bars: 50 μm. Source data are provided (see ‘Data availability’).

Article Snippet: A dominant negative form of the human Rab7A (T22N) was obtained from plasmid DsRed-rab7 DN (a gift from Richard Pagano, Addgene plasmid #12662 ).

Techniques: Derivative Assay, Expressing, Dominant Negative Mutation, Membrane, Functional Assay, Immunostaining, Fluorescence