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: PLoS ONE

Article Title: Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

doi: 10.1371/journal.pone.0048853

Figure Lengend Snippet: Representative confocal micrographs of Vero cells infected with ASFV and immunostained for viral capsid proteins p72 and pE120R (shown in red), and in green, EE marker EEA1 ( A ), MVB marker CD63 ( B ), LE marker Rab7 ( C ) and LY marker Lamp1 ( D ) at 15 minutes postinfection (mpi). Scale bars, 10 µm. Cells were infected at a moi of 10 pfu/cell and adsorption was maintained at 4°C for 90 min. Unbound virus was then washed, cells were shifted to 37°C and infection was allowed to progress for indicated times. ( E ) Percentages of colocalization events of p72 capsid protein with EE or LE marker are expressed as means and relativized to the total cell-associated virus particles per individual cell at each time point in 10 cells in duplicates. ( F ) Percentages of colocalization events of p150 inner core protein with LE marker expressed as means and relativized to the total cell-associated virus particles per individual cell at each time point in 10 cells in duplicates. ( G ) Representative confocal micrograph of the colocalization of viral cores with Rab7 positive endosomes. Nuclei were stained with TOPRO3. ( G1–4 ) Detail of colocalization between viral cores and LE in high magnification of the boxed areas in (G). ( H ) Colocalization of viral core protein p150 with Lamp1 marker.

Article Snippet: Wild-type GFP-tagged human Rab7 and dominant-negative mutant (Rab7 T22N) plasmid constructs cloned as N-terminal GFP fusions in the pGreenLantern vector (Gibco-BRL, Grand Island NY, USA) were kindly provided by Dr, Craig Roy, Yale University, USA.

Techniques: Infection, Marker, Adsorption, Staining

Journal: PLoS ONE

Article Title: Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

doi: 10.1371/journal.pone.0048853

Figure Lengend Snippet: ( A ) Early viral protein p30 expression determined at 8 hpi by Western blotting with specific antibodies, quantified and normalized to protein load control values. Acid pH requirement was evidenced by the effect of lysosomotropic drug addition at any time point within the first hpi but not thereafter. ( B ) Representative confocal micrograph of Baf-pretreated cells fixed after 3 hpi and immunostained for major viral capsid protein p72 (red) and LE marker Rab7 (green); Bar 10 µm. Detail of colocalization between viral capsids and LEs in Baf-treated cells; Insets are magnifications of the boxed areas in the previous image, bar 1 µm. ( C ) Quantification of colocalization events relativized to the total number of cell-associated virions per individual cell, performed in 130 virions and expressed as means and standard deviations from two independent experiments. Asterisks denote statistically significant differences (*** P <0.001).

Article Snippet: Wild-type GFP-tagged human Rab7 and dominant-negative mutant (Rab7 T22N) plasmid constructs cloned as N-terminal GFP fusions in the pGreenLantern vector (Gibco-BRL, Grand Island NY, USA) were kindly provided by Dr, Craig Roy, Yale University, USA.

Techniques: Expressing, Western Blot, Marker

Journal: PLoS ONE

Article Title: Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

doi: 10.1371/journal.pone.0048853

Figure Lengend Snippet: ( A ) Representative FACS profiles obtained during sorter analysis of COS-7 cells transfected with GFP-Rab7-wild type (Rab7 WT) and dominant negative mutant (GFP-Rab7-DN, T22N). R3 represents transfected cells expressing GFP to be sorted. ( B ) Representative confocal micrographs of transfected, sorted cells after isolation, infected with ASFV at a moi of 1 for 24 hpi and immunostained for major viral capsid protein p72 (red). Percentages of transfected infected cells decreased from 43.5% in cells expressing Rab7 WT to 1.65% in cells expressing Rab7 DN. Bar 25 µm.

Article Snippet: Wild-type GFP-tagged human Rab7 and dominant-negative mutant (Rab7 T22N) plasmid constructs cloned as N-terminal GFP fusions in the pGreenLantern vector (Gibco-BRL, Grand Island NY, USA) were kindly provided by Dr, Craig Roy, Yale University, USA.

Techniques: Transfection, Dominant Negative Mutation, Expressing, Isolation, Infection

Journal: PLoS ONE

Article Title: Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

doi: 10.1371/journal.pone.0048853

Figure Lengend Snippet: ( A ) Quantification of virus production in cells untreated, treated with 1 µM PIKfyve inhibitor YM201636 or treated with DMSO. Data are expressed as virus titers and are means ± SD from three independent experiments. Asterisks denote statistically significant differences ** P <0.01; * P <0.05 ( B ) Infected cell numbers in cells treated with PIKfyve inhibitor (YM201636) at several time points or an equivalent volume of DMSO. Data are expressed as the number of infected cells at 6 hpi (moi of 1 pfu/cell) from 20 random fields and are means ± SD from two independent experiments. Asterisks denote statistically significant differences (*** P <0.001 and ** P <0.01). ( C ) Representative confocal micrographs of infected and non-infected PIKfyve-treated cells, immunostained for Rab7 (green) and viral protein p72 (red). The characteristic phenotype of cytoplasmic vacuoles due to impaired endosome fusion was readily found in uninfected cells. Infected cells are recognized in the image as those harboring viral factories in red and lacked cytoplasmic vacuolization phenotype. Bar 10 µm.

Article Snippet: Wild-type GFP-tagged human Rab7 and dominant-negative mutant (Rab7 T22N) plasmid constructs cloned as N-terminal GFP fusions in the pGreenLantern vector (Gibco-BRL, Grand Island NY, USA) were kindly provided by Dr, Craig Roy, Yale University, USA.

Techniques: Infection

Journal: PLoS ONE

Article Title: Endosomal Maturation, Rab7 GTPase and Phosphoinositides in African Swine Fever Virus Entry

doi: 10.1371/journal.pone.0048853

Figure Lengend Snippet: ASFV enters the host cell by clathrin-coated vesicles (CCVs) from clathrin-coated pits (CCPs) and clathrin molecules are recycled to the plasma membrane (PM) as the virus progresses to the endosomal pathway. First, virions gain access to EEs from PM. The EE compartment is characterized by the presence of Rab5 and EEA1. ASFV is then directed from the vacuolar domain of the EE to the acidic late compartments. Subsequently, the virions reach CD63 enriched membranes of MVBs. Under the acid intraluminal pH of these endosomes, viral capsid would be degraded and viral cores would reach LE which depends on the presence of Rab7. At this stage, viral cores could egress to the cytosol to reach their replication site at the perinuclear area. In this process, the PIs composition of the endosomal membrane seemed to be crucial. PtdIns3P is synthesized by PI3K and this process is inhibited by PI3K inhibitor wortmannin and PtdIns(3,5)P 2 , which is synthesized by the enzyme PIKfyve, a process blocked by the inhibitor YM201636. These PIs interconversions on the endosomal membrane are necessary for a successful infection.

Article Snippet: Wild-type GFP-tagged human Rab7 and dominant-negative mutant (Rab7 T22N) plasmid constructs cloned as N-terminal GFP fusions in the pGreenLantern vector (Gibco-BRL, Grand Island NY, USA) were kindly provided by Dr, Craig Roy, Yale University, USA.

Techniques: Synthesized, Infection

Journal: Molecular and Cellular Biology

Article Title: Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux

doi: 10.1128/MCB.00392-17

Figure Lengend Snippet: C18orf8/RMC1 is required for endolysosomal size control and autophagic flux. (A) HeLa cells stably expressing RMC1-FLAG-HA were grown on glass coverslips, fixed, and immunostained as indicated. (B) HeLa cells were transfected with control or RMC1 siRNA pools; at 72 h posttransfection, cells were harvested for immunoblot analysis with the indicated antibodies. (C) Cells treated with siRNAs as for panel B were grown on glass coverslips, fixed, and immunostained with the late endosomal marker anti-CD63; maximum-intensity projections of z-stacks are shown, and scale bars represent 20 μm. (D) Cells treated with siRNAs as for panel B were fixed and analyzed by TEM. (E) HeLa cells were treated with individual or combined siRNAs as indicated for 72 h, followed by immunoblot analysis to evaluate depletion of RMC1 and accumulation of p62 protein. (F) Quantification of p62 levels shown in panel E for two biological replicate experiments. Error bars represent standard deviation, and significance was determined by one-way ANOVA with post hoc testing (Dunnett's multiple-comparison test). *, P < 0.05; **, P < 0.01. (G) HeLa cells were grown on glass coverslips, fixed, and immunostained with anti-CD63 and LC3B; representative maximum-intensity projections are shown, and scale bars represent 20 μm. (H) Quantification of basal LC3B punctum number in cells stained as for panel G. Data represent two pooled biological experiments, and error bars represent standard deviation. (I and J) HeLa cells treated with control or RMC1 siRNAs (as for panel E) were treated with Cell Light RFP-GFP-LC3B for 16 h, followed by incubation in nutrient-replete DMEM (I) or starvation medium (HBSS) (J) for 1 h. Autophagic flux was monitored by confocal microscopy; single z-sections are shown for each channel, and scale bars represent 20 μm. (K and L) Quantification of average percentage of RFP-GFP-positive and RFP-only puncta per cell as shown in panels I and J. Data represent two pooled biological experiments, error bars represent standard deviation of the mean, and significance was determined by one-way ANOVA with post hoc testing (Dunnett's multiple-comparison test). ****, P < 0.0001. (M) Left panel, HeLa cells were grown on glass coverslips in nutrient-replete medium, fixed, and stained with endogenous antibodies against RAB7 and CD63. Individual representative z-sections for each channel are shown, and scale bars represent 20 μm. Right panel, intensity profiles of a 20-μm line segment drawn across the z-section shown for each channel. Overlap of 488 (RAB7) and 561 (CD63) fluorescence intensity profiles (arbitrary units [AU]) indicates colocalization of RAB7 with the late endosomal marker CD63.

Article Snippet: GFP-RAB7 WT , GFP-RAB7 Q67L , and GFP-RAB7 T22N (dominant negative) were gifts from Qing Zhong (Addgene plasmids 28047, 28049, and 28048, respectively) ( 62 ).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Marker, Standard Deviation, Staining, Incubation, Confocal Microscopy, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Systematic Analysis of Human Cells Lacking ATG8 Proteins Uncovers Roles for GABARAPs and the CCZ1/MON1 Regulator C18orf8/RMC1 in Macroautophagic and Selective Autophagic Flux

doi: 10.1128/MCB.00392-17

Figure Lengend Snippet: Identification of C18orf8/RMC1 as a member of the CCZ1-MON1 GEF complex for RAB7. (A) SILAC mass spectra of heavy (control) and light (ΔRAP-enriched) C18orf8/RMC1 peptides identified in autophagosomal proteomics; K represents the heavy-labeled lysine residue. (B) Top panel, predicted domain structure of RMC1. Bottom panel, Map of high-confidence interacting proteins (HCIPs) identified in reciprocal affinity purification-mass spectrometry (AP-MS) experiments with RMC1-FLAG-HA, FLAG-HA-CCZ1, or MON1A-MYC-FLAG bait proteins expressed in 293T cells. The line thickness correlates with the number of peptides observed in each interaction. (C) GFP-CCZ1 associates with RMC1-FLAG-HA. 293T cells stably expressing RMC1-FLAG-HA were transiently transfected with FLAG-HA-GFP control or GFP-CCZ1, followed by GFP-TRAP affinity purification. Note that RMC1-FLAG-HA associates with GFP-CCZ1 but not the FLAG-HA-GFP control. (D and E) Size exclusion chromatography and AP-MS of RMC1-containing complexes. 293T extracts stably expressing RMC1-FLAG-HA were separated by size exclusion chromatography; estimated molecular mass (MM) standards corresponding to fraction number are shown. Fractions containing RMC1 and RAB7 signals were subjected to FLAG AP-MS to identify complex members. (F) GFP-RAB7 associates with endogenous RMC1. 293T cells were transiently transfected with FLAG-HA-GFP control, GFP-RAB7WT, GFP-RAB7Q67L (constitutively active), or GFP-RAB7T22N (dominant negative), followed by GFP-TRAP affinity purification and immunoblot analysis as indicated.

Article Snippet: GFP-RAB7 WT , GFP-RAB7 Q67L , and GFP-RAB7 T22N (dominant negative) were gifts from Qing Zhong (Addgene plasmids 28047, 28049, and 28048, respectively) ( 62 ).

Techniques: Labeling, Affinity Purification, Mass Spectrometry, Stable Transfection, Expressing, Transfection, Size-exclusion Chromatography, Dominant Negative Mutation, Western Blot