Journal: Oncology Letters

Article Title: Tumor-suppressive microRNA-452 inhibits migration and invasion of breast cancer cells by directly targeting RAB11A

doi: 10.3892/ol.2017.6426

Figure Lengend Snippet: miR-452 mimic sequence alongside RAB11A-3′-UTR-WT and RAB11A-3′-UTR-Mut mRNA sequences, including the miR-452-binding site of the WT sequence, and the equivalent area from the Mut sequence. miR-452, microRNA-452; 3′-UTR, 3′-untranslated region; WT, wild type; Mut, mutant.

Article Snippet: Luciferase reporter assay Plasmids containing the wild-type RAB11A 3′-UTR (PmirGLO-RAB11A-3′-UTR-WT) or a mutant RAB11A 3′-UTR (PmirGLO-RAB11A-3′-UTR-Mut; ) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: Oncology Letters

Article Title: Tumor-suppressive microRNA-452 inhibits migration and invasion of breast cancer cells by directly targeting RAB11A

doi: 10.3892/ol.2017.6426

Figure Lengend Snippet: miR-452 directly targets RAB11A in breast cancer cells. The relative RAB11A (A) mRNA and (B) protein expression levels were significantly reduced in the miR-452 mimic-transfected breast cancer cells, compared with miR-NC-transfected cells. (C) A luciferase reporter assay was performed on HEK293T cells co-transfected with an miR-452 mimic or miR-NC, and PmirGLO-RAB11A-3′-UTR-WT or PmirGLO-RAB11A-3′-UTR-Mut, revealing that miR-452 decreased activity only in cells transfected with the WT RAB11A 3′-UTR. Data are presented as the mean ± standard deviation. *P<0.05 vs. corresponding miR-NC group. miR-452, microRNA-452; miR-NC, microRNA negative control; 3′-UTR, 3′-untranslated region; WT, wild type; Mut, mutant.

Article Snippet: Luciferase reporter assay Plasmids containing the wild-type RAB11A 3′-UTR (PmirGLO-RAB11A-3′-UTR-WT) or a mutant RAB11A 3′-UTR (PmirGLO-RAB11A-3′-UTR-Mut; ) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Expressing, Transfection, Luciferase, Reporter Assay, Activity Assay, Standard Deviation, Negative Control, Mutagenesis

Journal: Oncology Letters

Article Title: Tumor-suppressive microRNA-452 inhibits migration and invasion of breast cancer cells by directly targeting RAB11A

doi: 10.3892/ol.2017.6426

Figure Lengend Snippet: Inhibition of RAB11A inhibits migration and invasion of MCF-7 and MDA-MB-231 breast cancer cells. (A) Compared with siR-NC, RAB11A siRNA decreased RAB11A protein expression in breast cancer cells. (B) Transwell migration and (C) Transwell invasion assays were performed, revealing that RAB11A knockdown decreased breast cancer cell migration and invasion capacities. Data are presented as the mean ± standard deviation. *P<0.05 vs. corresponding siR-NC group. siRNA, small interfering RNA; siR-NC, negative control siRNA.

Article Snippet: Luciferase reporter assay Plasmids containing the wild-type RAB11A 3′-UTR (PmirGLO-RAB11A-3′-UTR-WT) or a mutant RAB11A 3′-UTR (PmirGLO-RAB11A-3′-UTR-Mut; ) were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China).

Techniques: Inhibition, Migration, Expressing, Knockdown, Standard Deviation, Small Interfering RNA, Negative Control

Journal: PLoS Biology

Article Title: Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

doi: 10.1371/journal.pbio.1002142

Figure Lengend Snippet: (A) Mammary gland sections stained for E-cadherin and PAR3 or aPKC. Arrows indicate cytoplasmic accumulation and impaired localization of PAR3 and aPKC to the apical surface; asterisks indicate small lumens. (B) Mammary gland sections stained for keratin 5 (K5) and GM130. (C) Percentage of LCs showing ribbon-like and fragmented localization of GM130 (control: n = 4 mice; mutant: n = 4 mice). (D) Mammary gland sections stained for HTT 4C8 and PAR3 or aPKC. (E) HTT/PAR3/PAR6/aPKC/RAB11A complexes were immunoprecipitated from MCF-10A cells. Mouse IgG (mIgG) was used as a negative control. The immunoprecipitates (IP) were analyzed by western blotting. All scale bars, 10 μm. Error bars, SEM. *** p <0.001. Complete statistical analyses with number of measures are detailed in .

Article Snippet: GFP-RAB11A wild-type (WT), dominant-negative (S25N), and constitutively active (Q70L) (referred to herein as RAB11A WT , RAB11A S25N and RAB11A Q70L , respectively) were obtained from B. Goud (Institut Curie, France).

Techniques: Staining, Control, Mutagenesis, Immunoprecipitation, Negative Control, Western Blot

Journal: PLoS Biology

Article Title: Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

doi: 10.1371/journal.pbio.1002142

Figure Lengend Snippet: (A) Mammary gland section stained for HTT 4C8 and RAB11A. (B) Mammary gland section stained for E-cadherin and RAB11A. (C) Twenty-four–hour and four-day MDCK 3-D cultures stained for HTT 4C8 and RAB11A. Colocalization of HTT and RAB11A is displayed in yellow (merge). (D) Twenty-four–hour and four-day MDCK 3-D cultures stained for RAB11A. HTTFL is tagged with mCherry and fluorescence is displayed in magenta and the colocalization of RAB11A and HTTFL appears in white. (E) Twenty-four–hour and four-day MDCK 3-D cultures transfected with RAB11A WT , RAB11A Q70L or RAB11A S22N , stained for PAR3. RAB11 is tagged with GFP and fluorescence is displayed in magenta. The colocalization of aPKC and RAB11A appears in white. (F) Representative line-scan analysis (relative fluorescence intensity; at least 20 cells were analyzed per condition). (G) Percentage of acini with normal lumen. (H) Quantification of acini size. (G and H) Control+RAB11A WT : n = 59 acini, Control+RAB11A Q70L : n = 54 acini, Control+RAB11A S22N : n = 66 acini, shHTT2+RAB11A WT : n = 60 acini, shHTT2+RAB11A Q70L : n = 72 acini, shHTT2+RAB11A S22N : n = 93 acini. (I) FM64-4 4-day MDCK 3-D structures were video recorded. Maximum intensity and z projections are shown. All scale bars, 10 μm. Error bars, SEM. ** p <0.01; *** p <0.001. Complete statistical analyses with number of measures are detailed in .

Article Snippet: GFP-RAB11A wild-type (WT), dominant-negative (S25N), and constitutively active (Q70L) (referred to herein as RAB11A WT , RAB11A S25N and RAB11A Q70L , respectively) were obtained from B. Goud (Institut Curie, France).

Techniques: Staining, Fluorescence, Transfection, Control

Journal: PLoS Biology

Article Title: Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

doi: 10.1371/journal.pbio.1002142

Figure Lengend Snippet: During epithelial morphogenesis, HTT modulates the activation of RAB11A (1). HTT-RAB11A forms a complex with PAR3-aPKC, which may be recruited to HTT-kinesin 1 apical vesicles (2). HTT coordinates apical recycling of PAR3-aPKC vesicles (3). PAR3-aPKC accumulation at the pre-apical patches (PAP) (4) triggers the expansion of the apical membrane, leading to the formation of a central lumen (5).

Article Snippet: GFP-RAB11A wild-type (WT), dominant-negative (S25N), and constitutively active (Q70L) (referred to herein as RAB11A WT , RAB11A S25N and RAB11A Q70L , respectively) were obtained from B. Goud (Institut Curie, France).

Techniques: Activation Assay, Membrane

Journal: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: A Primary hippocampal neurons (DIV6+4) were transfected with myc‐tagged TDP‐43 wild type or a mutant lacking the nuclear localization signal (ΔNLS) or an empty vector control. Immunostaining with the indicated antibodies and DAPI to label nuclei. Scale bar represents 50 μm. B Primary hippocampal neurons (DIV6+3) were transfected with either TDP‐43 wild type, TDP‐43ΔNLS, or an empty vector control together with GFP‐RAB11 to visualize recycling endosomes and analyzed as in Fig . C, D Quantitative analysis of recycling endosome movement (C) and vesicle number (D). Mean ± s.e.m., n = 4, one‐way ANOVA with Tukey's post‐test: * P < 0.05, ** P < 0.01.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Transfection, Mutagenesis, Plasmid Preparation, Control, Immunostaining

Journal: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: A Primary hippocampal neurons (DIV6+3) were transfected with shCtrl and empty vector control or shTDP with either empty vector control, TDP‐43 wild type, or the ΔNLS variant together with GFP‐RAB11 to visualize recycling endosomes. Representative dendrite segments and kymographs of GFP‐RAB11 vesicle movement. Scale bar represents 12 s (vertical) and 25 μm (horizontal). B, C Quantitative analysis of vesicle motility (B) and the number (C) from kymographs in (A). Mean ± s.e.m., n = 5, one‐way ANOVA: * P < 0.05.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Transfection, Plasmid Preparation, Control, Variant Assay

Journal: The EMBO Journal

Article Title: TDP‐43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons

doi: 10.15252/embj.201694221

Figure Lengend Snippet: A, B Luciferase assay to analyze transcriptional regulation of VPS4B by TDP‐43. (A) Reporter construct expressing Renilla luciferase (R‐Luc) driven by a VPS4B promoter fragment and firefly luciferase (FF) under the TK promoter. (B) HEK293 cells were transfected with shRNAs targeting human TDP‐43, a control shRNA, TDP‐43 wild type, or empty vector control together with the luciferase reporter containing the rat VPS4B promoter. VPS4B promoter‐driven Renilla luciferase activity was normalized to TK promoter‐driven firefly luciferase. Quantification from six independent experiments. C, D ChIP assay to analyze binding of TDP‐43 to VPS4B promoter region in rat cortical neurons, HEK293 cells, and human brain tissue. PCR from input, negative control, and TDP‐43 immunoprecipitates. Signal intensities from at least three independent experiments for each condition were quantified by densitometry and depicted as percentage of input. E–H Analysis of VPS4B in the tissues of seven patients with the neuropathological diagnosis of FTLD/ALS‐TDP and five healthy controls. (E, G) Immunofluorescence with the indicated antibodies in the superior frontal gyrus or spinal cord. Scale bar represents 50 μm. (F, H) Quantification of cellular VPS4B levels. At least 50 cells per case were analyzed. Data information: Mean ± s.e.m., unpaired, two‐tailed t ‐test: * P < 0.05, ** P < 0.01. Source data are available online for this figure.

Article Snippet: Human RAB4 (Addgene #54943, M. Davidson unpublished), RAB5 (Lang et al , ), RAB7 and RAB11a wild type and S25N (Addgene #12605, 12674, 12678, Richard Pagano (Choudhury et al , )) were expressed from pEGFP‐C1 vector.

Techniques: Luciferase, Construct, Expressing, Transfection, Control, shRNA, Plasmid Preparation, Activity Assay, Binding Assay, Negative Control, Biomarker Discovery, Immunofluorescence, Two Tailed Test