Journal: International Journal of Molecular Sciences

Article Title: Gastric Cancer Extracellular Vesicles Tune the Migration and Invasion of Epithelial and Mesenchymal Cells in a Histotype-Dependent Manner

doi: 10.3390/ijms20112608

Figure Lengend Snippet: Scheme illustrating the experimental design of the study. Four distinct gastric cancer (GC) cell lines (MKN74, MKN45, Kato III, and IPA220) were used as donor cells of extracellular vesicles (EVs), which were isolated by differential centrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and imaging flow cytometry. Epithelial (red) and mesenchymal (green) cells were used as recipients of GC-EVs and submitted to distinct functional assays.

Article Snippet: Human GC cell lines MKN74, MKN45, Kato III (ATCC), and IPA220 (established at Ipatimup [ ]) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France) and 1% PS (Penicillin-Streptomycin, Thermo Fisher Scientific) at 37 °C in 5% CO 2 humidified atmosphere.

Techniques: Isolation, Centrifugation, Transmission Assay, Electron Microscopy, Imaging, Flow Cytometry, Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Gastric Cancer Extracellular Vesicles Tune the Migration and Invasion of Epithelial and Mesenchymal Cells in a Histotype-Dependent Manner

doi: 10.3390/ijms20112608

Figure Lengend Snippet: Characterization of EVs secreted by MKN74, MKN45, Kato III, and IPA220 GC cell lines. ( A ) Representative electron microscopy images of EVs isolated from GC cells. Scale bars: 200 nm; ( B ) NTA of isolated EVs with mode size distribution (left) and particle concentration (right). Graphs represent the mean ± standard deviation of at least 14 biological replicates; ( C ) detection of CD9, CD81, Flotillin-1, and Cytochrome C (negative control) by imaging flow cytometry. Distribution and representative images of the intensity of fluorescence detected for each marker in three biological replicates. Bright-field images (BF) show beads to which EVs were coupled, fluorescence images (AF488) show EVs labeled with specific markers, and merged images (M) show labeled EVs coupled to beads.

Article Snippet: Human GC cell lines MKN74, MKN45, Kato III (ATCC), and IPA220 (established at Ipatimup [ ]) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France) and 1% PS (Penicillin-Streptomycin, Thermo Fisher Scientific) at 37 °C in 5% CO 2 humidified atmosphere.

Techniques: Electron Microscopy, Isolation, Concentration Assay, Standard Deviation, Negative Control, Imaging, Flow Cytometry, Fluorescence, Marker, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Gastric Cancer Extracellular Vesicles Tune the Migration and Invasion of Epithelial and Mesenchymal Cells in a Histotype-Dependent Manner

doi: 10.3390/ijms20112608

Figure Lengend Snippet: GC-EVs impair the migration of epithelial and mesenchymal cells. ( A ) Time-lapse microscopic images illustrating the migration of non-treated (control) and treated epithelial and mesenchymal cells, at distinct time-points; ( B , C ) the migration rate of non-treated (control) and treated epithelial and mesenchymal cells was assessed by time-lapse microscopy; ( B ) graphs represent the mean percentage of wound closure ± standard deviation of three independent experiments; ( C ) graphs represent the mean of normalized wound closure ± standard deviation of three independent experiments; the data was normalized to the control cells at each time-point (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, and two-way ANOVA with Dunnett’s multiple comparisons test; blue and orange asterisks correspond to E and M cells treated with IPA220-EVs or Kato III-EVs, respectively.

Article Snippet: Human GC cell lines MKN74, MKN45, Kato III (ATCC), and IPA220 (established at Ipatimup [ ]) were cultured in RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, France) and 1% PS (Penicillin-Streptomycin, Thermo Fisher Scientific) at 37 °C in 5% CO 2 humidified atmosphere.

Techniques: Migration, Control, Time-lapse Microscopy, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Human effector memory (EM) CD8 + T cell subsets differentially express Kv1.3 and KCa3.1. (A–C) Peripheral blood mononuclear cells from healthy individuals were stained with antibodies (Abs) to CD8, C-C chemokine receptor type 7 (CCR7), and interleukin (IL)-7Rα and sorted into IL-7Rα high and IL-7Rα low subsets of EM CD8 + T cell using a BD FACSAria ® . (A) Freshly sorted CD8 + T cell subsets were stimulated for 24 h with anti-CD3/CD28 Abs, and their KCa3.1 (IL-7Rα high , n = 23; IL-7Rα low , n = 23) and Kv1.3 (IL-7Rα high , n = 18; IL-7Rα low , n = 19) current components underlying the total current–voltage (I–V) curve were measured as follows: (i) the maximum inactivation of Kv1.3 was induced using depolarized holding voltage (−10 mV); (ii) a reverse ramp-like pulse from 60 to −120 mV was applied; and (iii) the obtained I–V curve for KCa3.1 was subtracted from the initial I–V curve obtained using a forward ramp-like pulse in the whole-cell configuration for the Kv1.3 current. (B) KCa3.1 was activated by subsequently adding 50 µM 1-ethyl-2-benzimidazolinone (1-EBIO) to verify the presence of functional KCa3.1 in IL-7Rα high ( n = 8) and IL-7Rα low ( n = 10) EM CD8 + T cells. (C) Cells were treated with a phosphatidylinositol 3-kinase inhibitor (10 µM LY294002) in IL-7Rα high ( n = 7) and IL-7Rα low ( n = 8) EM CD8 + T cells. (D) The current components of KCa3.1 (IL-7Rα high , n = 17; IL-7Rα low , n = 16) and Kv1.3 (IL-7Rα high , n = 18; IL-7Rα low , n = 19) were measured in IL-2 reversed IL-7Rα high and IL-7Rα low EM CD8 + T cells. The results were obtained by combining data from two independent experiments using two different donors. Bars and error bars represent the mean ± SEM (A–D) , and p -values were obtained using the two-tailed Student’s t -test (A,B,D) or the analysis of variance followed by Tukey’s post hoc subgroup analysis (C) .

Article Snippet: Tissue sections (7 μm) were fixed in 4% paraformaldehyde, blocked with a blocking buffer (5% goat serum and 5% BSA in PBS) for 30 min at room temperature, and stained with purified anti-human perforin Ab (Mabtech, Nacka Strand, Sweden), Alexa647-conjugated anti-human CD8 Ab (BD Biosciences), biotin-conjugated anti-human IL-7Rα Ab (13-1278, eBioscience), Streptavidin-Cy3 (Life Technologies), and Alexa488-conjugated anti-mouse IgG Ab (Life Technologies) at 4°C overnight.

Techniques: Staining, Functional Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Requirement of Kv1.3 for effector memory (EM) CD8 + T cell proliferation and interleukin (IL)-2 production. (A) Freshly sorted IL-7Rα high EM CD8 + T cells were labeled with carboxyfluorescein diacetate (CFSE) and stimulated for 6 days with anti-CD3/CD28 antibodies (Abs) in the presence or absence of potassium channel inhibitors such as TRAM-34 (KCa3.1 inhibitor, 5 µM) and margatoxin (Kv1.3 inhibitor, 5 nM), and their proliferation was measured by flow cytometry. Representative histograms and a quantification graph showing proliferating cells are shown. (B) Quantification of cytokines in culture supernatants from IL-7Rα high EM CD8 + T cells that were stimulated for 24 h with anti-CD3/CD28 Abs in the presence or absence of potassium channel inhibitors using a multiplex cytokine assay. Bars indicate the mean. The results are representative data from two or three independent experiments. Bars represent the mean, and p -values were obtained using the paired two-tailed Student’s t -test.

Article Snippet: Tissue sections (7 μm) were fixed in 4% paraformaldehyde, blocked with a blocking buffer (5% goat serum and 5% BSA in PBS) for 30 min at room temperature, and stained with purified anti-human perforin Ab (Mabtech, Nacka Strand, Sweden), Alexa647-conjugated anti-human CD8 Ab (BD Biosciences), biotin-conjugated anti-human IL-7Rα Ab (13-1278, eBioscience), Streptavidin-Cy3 (Life Technologies), and Alexa488-conjugated anti-mouse IgG Ab (Life Technologies) at 4°C overnight.

Techniques: Labeling, Flow Cytometry, Multiplex Assay, Cytokine Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: KCa3.1 mediates the motility of effector memory (EM) CD8 + T cells. The migration of EM CD8 + T cells under agarose gel confinement was recorded by time-lapse microscopy (objective 40×; Zeiss Axio Observer Z1; numerical aperture = 1.3; Plan-Neofluar) and analyzed using ImageJ and Metlab. Flat polyurethane acrylate (PUA) surfaces were coated with 10 µg/mL intercellular adhesion molecule 1 (ICAM-1) and/or 2 µg/mL stromal cell-derived factor (SDF)-1α. (A) The effect of ICAM-1 and/or SDF-1α on the mean velocity ( V mean ) of interleukin (IL)-7Rα high and IL-7Rα low EM CD8 + T cells on flat PUA surfaces. The surfaces containing >35 individual cells were analyzed. (B) IL-7Rα high EM CD8 + T cells were treated with either TRAM-34 (5 µM) or margatoxin (50 nM), and the motility of the drug-treated cells on ICAM-1 and/or SDF-1α-coated flat PUA surfaces was recorded by time-lapse microscopy. The surfaces containing >20 individual cells were analyzed. (C) The migration of IL-2-reversed IL-7Rα high and IL-7Rα low EM CD8 + T cells was analyzed on ICAM-1 and/or SDF-1α-coated flat PUA surfaces. The results are representative data from two independent experiments using two different donors. Bars represent the mean, and p -values were obtained using the unpaired two-tailed Student’s t -test.

Article Snippet: Tissue sections (7 μm) were fixed in 4% paraformaldehyde, blocked with a blocking buffer (5% goat serum and 5% BSA in PBS) for 30 min at room temperature, and stained with purified anti-human perforin Ab (Mabtech, Nacka Strand, Sweden), Alexa647-conjugated anti-human CD8 Ab (BD Biosciences), biotin-conjugated anti-human IL-7Rα Ab (13-1278, eBioscience), Streptavidin-Cy3 (Life Technologies), and Alexa488-conjugated anti-mouse IgG Ab (Life Technologies) at 4°C overnight.

Techniques: Migration, Agarose Gel Electrophoresis, Time-lapse Microscopy, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Decreased transendothelial migration of interleukin (IL)-7Rα low effector memory (EM) CD8 + T cells compared to that of IL-7Rα high EM CD8 + T cells. (A) freshly isolated or (B) IL-2-reversed IL-7Rα high and IL-7Rα low EM CD8 + T cells were added on top of human umbilical vein endothelial cell monolayers on the filters in the presence or absence of stromal cell-derived factor (SDF)-1α (100 ng/mL). Cells were allowed to migrate into the lower chambers or underneath the upper transwells for 20 h. Data are expressed as the number of cells that migrated across the filter. Results are representative data from two independent experiments from five to eight different donors. Bars represent the mean, and p -values were obtained using the unpaired two-tailed Student’s t -test.

Article Snippet: Tissue sections (7 μm) were fixed in 4% paraformaldehyde, blocked with a blocking buffer (5% goat serum and 5% BSA in PBS) for 30 min at room temperature, and stained with purified anti-human perforin Ab (Mabtech, Nacka Strand, Sweden), Alexa647-conjugated anti-human CD8 Ab (BD Biosciences), biotin-conjugated anti-human IL-7Rα Ab (13-1278, eBioscience), Streptavidin-Cy3 (Life Technologies), and Alexa488-conjugated anti-mouse IgG Ab (Life Technologies) at 4°C overnight.

Techniques: Migration, Isolation, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Interleukin (IL)-2 and IL-15 stimulation control the KCa3.1 activity in IL-7Rα low effector memory (EM) CD8 + T cells. (A,B) To measure the current components of cytokine-stimulated IL-7Rα high and IL-7Rα low EM CD8 + T cells, cells were stimulated for 3 days with anti-CD3/CD28 antibodies in the presence of IL-2 (20 IU/mL), IL-15 (5 ng/mL), or IL-4 (5 ng/mL), and their KCa3.1 (A) and Kv1.3 (B) current components (IL-7Rα high versus IL-7Rα low : n = 17 versus n = 15; n = 8 versus n = 7; or n = 6 versus n = 7, respectively) were measured as described in Figure . Bars and error bars represent the mean ± SEM, and p -values were obtained using the unpaired two-tailed Student’s t -test. (C) The migration of cytokine-stimulated IL-7Rα low EM CD8 + T cells was analyzed as described in Figure . The results are representative data from two independent experiments using two different donors. Bars represent the mean, and p -values were obtained using the unpaired two-tailed Student’s t -test.

Article Snippet: Tissue sections (7 μm) were fixed in 4% paraformaldehyde, blocked with a blocking buffer (5% goat serum and 5% BSA in PBS) for 30 min at room temperature, and stained with purified anti-human perforin Ab (Mabtech, Nacka Strand, Sweden), Alexa647-conjugated anti-human CD8 Ab (BD Biosciences), biotin-conjugated anti-human IL-7Rα Ab (13-1278, eBioscience), Streptavidin-Cy3 (Life Technologies), and Alexa488-conjugated anti-mouse IgG Ab (Life Technologies) at 4°C overnight.

Techniques: Control, Activity Assay, Two Tailed Test, Migration

Journal: Frontiers in Immunology

Article Title: Differentially Expressed Potassium Channels Are Associated with Function of Human Effector Memory CD8 + T Cells

doi: 10.3389/fimmu.2017.00859

Figure Lengend Snippet: Greater numbers of interleukin (IL)-7Rα high effector memory (EM) CD8 + T cells than IL-7Rα low EM CD8 + T cells in the skin. (A) Immunofluorescence staining (40×) of CD8 + T cells (green) from non-lesional (healthy, HC) or lesional atopic dermatitis skin. IL-7Rα + CD8 + T cells (upper panel) representing IL-7Rα high EM CD8 + T cells were stained with antibodies (Abs) to IL-7Rα (red); perforin + CD8 + T cells (lower panel) representing IL-7Rα low EM CD8 + T cells, were stained with Abs to perforin (red). The image of the box was magnified twice and placed to the right of each image. (B) A quantitative measurement of IL-7Rα + and perforin + CD8 + T cells (frequency and number per tissue) in panel (A) , representing IL-7Rα high and IL-7Rα low EM CD8 + T cells, respectively. Four images per slide were evaluated for quantification. Data are representative of four independent experiments. Bars represent the mean, and p -values were obtained using the Wilcoxon matched pairs test (for comparing frequency and number between the two CD8 + T cell subsets) and Mann–Whitney U test (for comparing IL-7Rα + /perforin + ratio between HC and dermatitis).

Article Snippet: Tissue sections (7 μm) were fixed in 4% paraformaldehyde, blocked with a blocking buffer (5% goat serum and 5% BSA in PBS) for 30 min at room temperature, and stained with purified anti-human perforin Ab (Mabtech, Nacka Strand, Sweden), Alexa647-conjugated anti-human CD8 Ab (BD Biosciences), biotin-conjugated anti-human IL-7Rα Ab (13-1278, eBioscience), Streptavidin-Cy3 (Life Technologies), and Alexa488-conjugated anti-mouse IgG Ab (Life Technologies) at 4°C overnight.

Techniques: Immunofluorescence, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a , b ) β4 protein (expression of the SCN4B gene) was analysed by immunohistochemistry on human breast tissue samples. ( a ) The expression of β4 protein was strong in epithelial cells of mammary acini (some examples are indicated by the black arrows), and not in non-epithelial cells of normal breast tissues. ( b ) In breast cancer tissue, the expression of β4 protein was strong in normal epithelial cells of mammary acini (black arrows), but significantly reduced in cancer cells (tumour area indicated by the red arrow, ‘T'). Scale bars, 50 μm.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Immunohistochemistry

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a – d ) SCN4B/ β4 protein expression was analysed by immunohistochemistry on breast tissue microarrays. Samples were stratified in ‘no staining', ‘weak staining' or ‘strong staining' groups. ( a ) Proportion of samples showing no (white), weak (gray) or strong (black) β4 staining in normal breast, compared with mammary hyperplasia/dysplasia and cancer (mixed grades) samples. The number of samples per condition is indicated in brackets. SCN4B /β4 protein staining was stronger in normal compared with cancer samples ( χ 2 , P <0.001), and in hyperplasia/dysplasia compared with cancer samples ( χ 2 , P <0.001). ( b ) SCN4B /β4 staining from indicated samples. Scale bars, 50 μm. ( c ) Proportion of samples showing no, weak or strong SCN4B /β4 staining in cancer samples, from grade I to III, and in lymph node metastases (LNM) samples. The number of samples per condition is indicated in brackets. β4 staining was stronger in grade I cancer samples compared with more advanced cancer samples (grades II, III and LNM) ( χ 2 , P <0.001). ( d ) Representative pictures of β4 staining from grade I, II and III ductal carcinoma samples. Scale bars, 50 μm. ( e ) Expression of the SCN4B gene in non-cancer ( n =29) and in invasive breast carcinoma tissues ( n =145) was analysed from The Cancer Genome Atlas (TCGA). RNA level is expressed as reads per kilobase per million (RPKM). Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. SCN4B gene was significantly downregulated in cancer tissues (Mann–Whitney rank sum test, MW, P <0.001). ( f , g ) Prognostic analyses of gene expression in breast cancers, performed using the Breast Cancer Gene-Expression Miner . ( f ) Kaplan–Meier Any Event (AE)-free survival analyses, performed on data pooled from cohorts for the expression of SCN4B gene ( n =1,024 patients). AE is defined as being metastatic relapse (MR) or patient death. A weak expression of SCN4B gene (≤ median of the pooled cohorts) was associated with a decrease in the AE-free survival ( P =0.0005). ( g ) Kaplan–Meier MR-free survival analyses were performed for the expression of SCN4B ( n =661 patients). A weak expression of SCN4B (≤ median of the pooled cohorts) was associated with a decrease in the MR-free survival ( P =0.0013). Cox results are displayed on the graph.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Immunohistochemistry, Staining, MANN-WHITNEY, Gene Expression

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) The expression of SCN4B gene was studied by RT–qPCR in human mammary epithelial non-cancer MCF-10A and cancer MCF7, MDA-MB-468, MDA-MB-435s and MDA-MB-231 cell lines. Results are expressed as relative to that of HPRT-1 gene ( n =7–12) and presented as mean values±s.e.m. *, significantly different from MCF-10A at P <0.05 (MW). ( b ) The expression of SCN4B /β4 protein was studied by densitometric analysis of western blot experiments in same cells as in a . Results are given as the amount of SCN4B /β4 protein relative to that of HSC70 ( n =5) and presented as mean values±s.e.m. *, significantly different from MCF-10A at P <0.05 (MW). The image on top shows a representative western blotting experiment. ( c ) The expression of SCNxB genes was analysed in MDA-MB-231-Luc cells by reverse transcription–PCR. Plasmids encoding human SCNxB genes were used as positive controls for PCR primers. ( d ) Representative western blotting experiments showing protein expression for β1 ( SCN1B ), β2 ( SCN2B ) and β4 ( SCN4B ) in MDA-MB-231-Luc cells. ( e ) Cells were transfected with scrambled siRNA (siCTL) or with siRNA directed against the expression of the SCN1B gene (si SCN1B ), the SCN2B gene (si SCN2B ) or the SCN4B gene (si SCN4B ). The efficacy of siRNA transfection was assessed by western blotting experiments 48 h after transfection. HSC70 was used as a control for sample loading. ( f ) Representative images of fixed and haematoxylin-stained MDA-MB-231-Luc cells on invasion inserts. Cancer cells were transfected with scrambled siCTL or with specific siRNA. Scale bars, 50 μm. ( g ) Summary of cancer cell invasiveness results ( n =8) for MDA-MB-231-Luc cells transfected with siCTL or si SCNxB . Results were expressed relative to siCTL and presented as mean values±s.e.m. ***, statistically different from siCTL at P <0.001 (Student's t -test). ( h ) Representative image of a zebrafish embryo injected in the yolk sac with MDA-MB-231-Luc cells stained with CM-Dil and showing sites of colonization. Scale bars, 500 μm. Below is a magnification of the highlighted region containing human cancer cells (see arrows) colonizing organs of the embryo. ( i ) Zebrafish colonization index of siCTL or si SCN4B cells. Numbers in brackets indicate the number of embryos examined for each condition, from three different experiments. Results are presented as mean values±s.e.m. **, statistically different from siCTL at P <0.01 (Student's t -test).

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Transfection, Control, Staining, Injection

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) Cancer cell invasiveness was assessed, using Matrigel-invasion chambers, from MDA-MB-231-Luc cells stably transfected with null-target shRNA (shCTL), SCN5A -targeting shRNA (sh SCN5A ) or SCN4B -targeting shRNA (sh SCN4B ), in the absence (−) or presence (+) of 30 μM TTX. The results from 8 to 16 independent experiments were expressed relative to control cells transfected with shCTL in the absence of TTX. ***, statistically different from shCTL at P <0.001 and #, statistically different from sh SCN4B in the absence of TTX at P <0.05. ( b ) Cancer cell invasiveness was likewise assessed in shCTL or sh SCN4B cells, transiently transfected with null-target siRNA (siCTL) or SCN5A -targeting siRNA (si SCN5A ). The results from 12 independent experiments were expressed relative to shCTL cells transfected with siCTL. ***, statistically different from the shCTL/siCTL condition at P <0.001 and #, statistically different from sh SCN4B /siCTL at P <0.05. ( c ) Cancer cell invasiveness was assessed in MDA-MB-231-Luc cells stably expressing the SCN5A -targeting shRNA (sh SCN5A ), not expressing the Na V 1.5 protein, and transiently transfected with null-target siRNA (siCTL) or SCN4B -targeting siRNA (si SCN4B ). This effect was assessed in the absence (−) or presence (+) of two TTX concentrations (3 or 30 μM), or 30 nM of the Na V 1.8 inhibitor A803467. The results from six independent experiments were expressed relative to sh SCN5A cells transfected with siCTL, in the absence of any Na V inhibitor. NS stands for no statistical difference and *** denotes a statistical difference from sh SCN5A /siCTL at P <0.001. ( d ) Cancer cell invasiveness was assessed using Matrigel-invasion chambers for MDA-MB-468 breast, H460 and A549 non-small-cell lung, and PC3 prostate cancer cells transfected with null-target siRNA (siCTL, black bar) or SCN4B -targeting siRNA (si SCN4B, red bars). Cancer cell lines known to express or not functional Na V channels are indicated as Na V + and Na V − , respectively. The results from 3 to 12 independent experiments were presented and are expressed relative to the results obtained with the same cells transfected with siCTL. *, different from siCTL at P <0.05 and *** at P <0.001. Statistics presented in this figure were performed using ANOVA for multiple group comparison ( a – c ) or Student's t -test ( d ). All results presented in this figure are mean values±s.e.m.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Stable Transfection, Transfection, shRNA, Control, Expressing, Functional Assay, Comparison

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) Sodium current ( I Na )–voltage relationships in shCTL (black squares, n =18) and in sh SCN4B (red circles, n =22) cells. There was a significant difference at P <0.001 between the two conditions in the voltage range between −40 and +40 mV. ( b ) Activation (filled circles)– and availability (filled squares )–voltage relationships in shCTL (black symbols) and sh SCN4B (red symbols) cells. ( c ) I Na peak and I Na persistent currents obtained from shCTL (black trace) and sh SCN4B (red trace) cells for a membrane depolarization from −100 to −30 mV. ( d ) Mean values±s.e.m. of I Na persistent currents obtained for a membrane depolarization from −100 to −30 mV from 18 shCTL and 21 sh SCN4B cells. NS, not statistically different. ( e ) Mean values±s.e.m. of I Na persistent/ I Na peak currents ratios obtained from 18 shCTL and 21 sh SCN4B cells. **, statistically different from shCTL at P <0.01. ( f ) Dose–response effect of TTX on the inhibition of I Na peak elicited by a membrane depolarization from −100 to −5 mV in shCTL (black squares, n =8–12) and in sh SCN4B (red circles, n =7–12) cells. Data were fitted with the Hill equation and IC 50 values were 2.02±0.10 and 2.24±0.11 μM for shCTL and sh SCN4B cells, respectively. ( g ) Intracellular pH measurements using the BCECF-AM probe, in NH 4 Cl-acidified shCTL (black trace) and sh SCN4B (red trace) cells in the absence of NaCl. NaCl (130 mM) was added at the time indicated (arrow). ( h ) H + efflux measurements after the addition of NaCl in conditions similar to g ( n =20). Results are expressed as mean values±s.e.m. NS, no statistical difference. ( i ) MDA-MB-231 shCTL or sh SCN4B cells were cultured on a Matrigel matrix containing DQ-Gelatin. A ‘Matrix-Focalized-degradation index' was calculated as being F-actin foci (red labelling, phalloidin-Alexa594) co-localized with focused proteolytic activities (green) ( n =442 cells for shCTL and 448 cells and sh SCN4B ). Results are expressed as mean values±s.e.m. NS, no statistical difference. ( j ) Representative pictures showing matrix degradation areas (green spots) and F-actin foci (red spots). Merging points (coloc), which appear as white pixels, were counted. Numbers of white pixels per cell were normalized to the mean value obtained in shCTL cells. Statistics were performed using Student's t -test.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Activation Assay, Membrane, Inhibition, Cell Culture

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) Cancer cell invasiveness was assessed using Matrigel-invasion chambers from shCTL or sh SCN4B MDA-MB-231 cells, in the absence (−) or presence of the protease inhibitors GM6001 (10 μM), leupeptin (200 μM) or E64 (100 μM). Results from three to seven independent experiments are presented and were expressed relative to shCTL cells in the absence of inhibitors. Results are expressed as mean values±s.e.m. *** denotes a statistical difference from the shCTL at P <0.001, and # indicates a statistical difference from sh SCN4B at P <0.05 (ANOVA). ( b ) Cancer cell migration of shCTL and sh SCN4B cells measured by time-lapse microscopy to track the movement of cells over 180 min, 1 frame per min ( n =20 representative cells in each condition). Distances are indicated in μm. ( c ) The speed of migration (in μm min −1 ) was analysed in shCTL and sh SCN4B cells from time-lapse experiments and results shown were obtained from 106 and 96 cells, respectively. *** denotes a statistical difference from the shCTL at P <0.001 (MW). ( d ) The track length of cell migration (in μm) was analysed over 180 min in shCTL and sh SCN4B cells from time-lapse experiments and results shown were obtained from 106 and 96 cells, respectively. *** denotes a statistical difference from the shCTL at P <0.001 (MW). ( e ) Three-dimension (3D) invasiveness of shCTL and sh SCN4B cells, embedded inside Matrigel, was measured by time-lapse microscopy to track the movement of cells over 48 h (1 frame per 30 min) in the absence (CTL) or presence of the MMP inhibitor GM6001 (10 μM) ( n =13 representative cells in each condition). Distances are indicated in μm. ( f ) The track length of 3D cell invasiveness (in μm) was analysed over 48 h in shCTL and sh SCN4B cells from time-lapse experiments and results shown were obtained from 30 cells in each condition. ** and *** denote statistical difference from the shCTL, CTL condition at P <0.01 and P <0.001, respectively. ## denotes a statistical difference from the sh SCN4B , CTL condition at P =0.002. § denotes a statistical difference from the shCTL, GM6001 condition at P =0.038 (Dunn's test). ( c , d and f ) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Migration, Time-lapse Microscopy

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) F-actin was stained with phalloidin-AlexaFluor594 in shCTL and sh SCN4B cells and a cell circularity index was calculated ( n =88 cells per condition). Results are expressed as mean values±s.e.m. *** P <0.001 from shCTL (Student's t -test). ( b ) Representative SEM micrographs of shCTL and sh SCN4B cells. Scale bars, 10 μm. ( c ) Number of filopodia-like structures per cell, counted from SEM pictures in shCTL and sh SCN4B cells ( n =60 and 66 cells, respectively). *** P <0.001 from shCTL (MW). ( d ) Number of blebs per cell, counted from SEM micrographs in shCTL and sh SCN4B cells ( n =82 cells per condition). *** P <0.001 from shCTL (MW). ( e ) Representative confocal micrographs of shCTL and sh SCN4B cells for which F-actin was stained with phalloidin-AlexaFluor488 (green) and nuclei with DAPI (blue), scale bar 20 μm. For enlargements images, scale bars are 5 μm. ( f ) Number of filopodia per cell, counted from confocal micrographs in shCTL and sh SCN4B cells ( n =46 and 66 cells, respectively). *** P <0.001 from shCTL (MW). ( g ) Length of filopodia, measured from confocal micrographs, in shCTL and sh SCN4B cells ( n =1,070 and 593 filopodia, respectively). ***, statistically different from shCTL at P <0.001 (MW). ( h ) SEM observations of sh SCN4B cell invasion 24 h after cells were seeded on a layer of Matrigel (4 mg ml −1 ). The coloured structure is the tip of the cell still observable above the Matrigel layer, while penetrating inside the matrix. Scale bar, 10 μm. ( i ) Western blots showing total and active GTP-bound forms of RhoA, Rac1 and Cdc42, pulled down by GST-RBD in shCTL and sh SCN4B cells. ( j ) Quantification of GTP-bound RhoGTPases (active), normalized to total protein level, and expressed relatively to that of shCTL ( n =5). **, statistically different from shCTL at P <0.01 and * at P <0.05 (MW). ( k ) shCTL or sh SCN4B cancer cell invasiveness, in the absence (−) or presence of blebbistatin (50 μM) ( n =3). Results are expressed as mean values±s.e.m. *** P <0.001 from shCTL, and ### P <0.001 from sh SCN4B (ANOVA). ( l ) Left panel, in situ proximity ligation assays showing a strong proximity between SCN4B /β4proteins and RhoA in shCTL cells (red dots, left panel) and the absence of any proximity signal in sh SCN4B cells (right panel). Scale bars, 50 μm. ( c , d , f , g , j ) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Staining, Western Blot, In Situ, Ligation

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) CTL, sh SCN4B and oe SCN4B cancer cell invasiveness, in the absence (−) or presence of TTX (30 μM), expressed relative to oeCTL cells in the absence of TTX ( n =6). Results are expressed as mean values±s.e.m. ***, different from CTL at P <0.001, ** at P <0.01. ### , different from sh SCN4B at P <0.001 (ANOVA). NS, no statistical difference. ( b ) Cancer cell invasiveness ( n =6) from oeCTL and oe SCN4B cells, in the absence (−) or presence of TTX (30 μM), expressed relative to oeCTL cells in the absence of TTX. Results are expressed as mean values±s.e.m. **, different from oeCTL at P <0.01. NS, no statistical difference (ANOVA). ( c ) I Na –voltage relationships in oeCTL (black squares, n =15) and oe SCN4B (green triangles, n =43) cells. There was a significant difference at P <0.05 between the two conditions in the voltage range between −45 and +45 mV. ( d ) Activation (filled circles)– and availability (filled squares)–voltage relationships obtained in the same oeCTL (black symbols) and oe SCN4B (green symbols) cells as in c . ( e ) Mean values±s.e.m. of I Na persistent currents obtained for a membrane depolarization from −100 to −30 mV from 15 oeCTL and 43 oe SCN4B cells. * P <0.05 from oeCTL (Student's t -test). ( f ) Mean values±s.e.m. of I Na persistent/ I Na peak current ratios in same conditions as in e . *** P =0.001 (Student's t -test). ( g ) oeCTL or oe SCN4B cells were cultured on a Matrigel-composed matrix containing DQ-Gelatin, and a ‘Matrix-Focalized-degradation index' was calculated ( n =77 and 69 cells for oeCTL and oe SCN4B , respectively). ***, statistically different from oeCTL at P <0.001 (MW). ( h ) Cell circularity index was calculated from oeCTL and oe SCN4B cells ( n =73 cells per condition). Results are expressed as mean values±s.e.m. ***, statistically different from oeCTL at P <0.001 (Student's t -test). ( i ) Speed of migration (μm min −1 ) of oeCTL and oe SCN4B cells analysed from time-lapse experiments ( n =47 per condition). ***, statistically different from oeCTL at P <0.001 (MW). ( j ) Western blots showing total and active GTP-bound forms of RhoA, Rac1, Cdc42, pulled down by GST- in oeCTL and oe SCN4B cells. ( k ) Quantification of GTP-bound RhoGTPases in oe SCN4B cells, normalized to its total protein level, and expressed relatively to that of oeCTL cells ( n =4). *, statistically different from the oeCTL at P <0.05. NS, no statistical difference (MW). ( g , i , k ) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Activation Assay, Membrane, Cell Culture, Migration, Western Blot

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) Cartoon showing the transmembrane structure of the β4 protein, encoded by the SCN4B gene. The extracellular domain contains an Ig-like structure. After introduction of synonymous nucleotide substitutions in the SCN4B sequence, we have generated a sequence that is not recognized by the small hairpin RNA targeting SCN4B expression. This sequence has been inserted into a pSec expression vector in order to overexpress the full-length β4 protein (called ‘Full-length') in sh SCN4B cells. Alternatively, we have also created truncated versions of the β4 protein: one containing a deletion of its intracellular C-terminus, from residue K185, and called ‘ΔC-ter', and one containing a deletion of its extracellular N-terminus up to residue T161, and called ‘ΔN-ter'. The nucleotide sequences were inserted into the pSec mammalian expression vector. ( b ) Cancer cell invasiveness was assessed using Matrigel-invasion chambers from shCTL and sh SCN4B , transfected with an empty expression vector (pSec), or transfected with ‘ΔN-ter', ‘ΔC-ter' or ‘Full-length' encoding sequences. Results are expressed as mean values±s.e.m. ### , statistically different from sh SCN4B /pSec at P <0.001 and ## at P <0.01. NS, not statistically different (ANOVA). ( c ) The speed of migration (in μm min −1 ) was analysed from time-lapse experiments with shCTL and sh SCN4B cells, transfected with an empty expression vector (pSec), or transfected with ‘ΔN-ter', ‘ΔC-ter' or ‘Full-length' encoding sequences, and results shown were obtained from 30 cells in each condition. ***, statistically different from shCTL at P <0.001. ### , statistically different from sh SCN4B /pSec at P <0.001. NS, not statistically different (Dunn's test). ( d ) Quantification of GTP-bound RhoA in sh SCN4B cells, transfected with empty vector (pSec), with ‘ΔN-ter', ‘ΔC-ter' or ‘Full-length' encoding sequences. The activity of GTP-bound (active) RhoAGTPase was normalized to its total protein level, and was expressed relatively to that of sh SCN4B /pSec cells ( n =3). *, statistically different from sh SCN4B /pSec at P <0.05 (MW). ( c , d ) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Sequencing, Generated, Expressing, Plasmid Preparation, Residue, Transfection, Migration, Activity Assay

Journal: Nature Communications

Article Title: SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer

doi: 10.1038/ncomms13648

Figure Lengend Snippet: ( a ) Top cartoon, transendothelial migration experiment. Bottom box plot, quantification of the number of shCTL, sh SCN4B or oe SCN4B cancer cells migrating through the endothelium (HUVEC monolayer) and the 8-μm pore-sized filter of the migration transwell, expressed as a relative number to shCTL (4 independent experiments). ( b ) Top cartoon, transendothelial invasion experiment. Bottom box plot, quantification of the number of shCTL, sh SCN4B or oe SCN4B cancer cells migrating through the extracellular matrix (matrigel) coating the 8-μm pore-sized filter of the invasion transwell, then endothelium (HUVEC monolayer), expressed as a relative number to shCTL (three independent experiments). ( c ) Bioluminescent imaging (BLI) performed in NMRI nude mice tail vein-injected with MDA-MB-231-Luc cells that do not express (sh SCN4B) , or which overexpress, the SCN4B protein (oe SCN4B ). Representative ex vivo lung BLI, after organ isolation, at completion of the study (ninth week after cell injection). ( d ) BLI quantification of excised lungs from mice injected with sh SCN4B cells ( n =7) and mice injected with oe SCN4B cells ( n =8) (MW). ( e ) Mean±s.e.m. in vivo BLI value of tumours (expressed in c.p.m.) as a function of time recorded in the whole body of mice. * denotes a statistical difference from the sh SCN4B group at P <0.05 (Student's t -test). ( f ) Representative bioluminescent images of mammary tumours in sh SCN4B and oe SCN4B experimental groups (23rd week after cell implantation). ( g ) Immunohistochemical analyses of primary mammary tumours obtained from same mice as in implanted with sh SCN4B cells (top image) or with oe SCN4B cells (bottom image). Slides were counterstained with haematoxylin (blue labelling), and incubated with anti-mouse SCN4B /β4 antibodies and immunohistochemistry was performed using the streptavidin-biotin-peroxidase method with diaminobenzidine as the chromogen (brown labelling). Scale bars, 100 μm. ( h ) Immunohistochemical analyses of lungs obtained from the same mice as in , implanted with human sh SCN4B cells (top image) and oe SCN4B cancer cells (bottom image). Slides were counterstained with haematoxylin (blue labelling), and human breast cancer cells were identified using anti-human cytokeratin 7 immunohistochemical (brown) labelling. Scale bars, 100 μm. ( a , b , d ) Box plots indicate the first quartile, the median and the third quartile; whiskers indicate minimum and maximum values; squares show the means. Error bars encompass 95% of data samples.

Article Snippet: MDA-MB-231-Luc human breast cancer cells were transfected with 20 nM siRNA targeting the expression of SCN1B (si SCN1B , sc-97849), SCN2B (si SCN2B , sc-96252), SCN4B (si SCN4B , sc-62982) or scrambled siRNA (siCTL, siRNA-A sc-37007), which were produced by Santa Cruz Biotechnology and were purchased from Tebu-Bio (France).

Techniques: Migration, Imaging, Injection, Ex Vivo, Isolation, In Vivo, Immunohistochemical staining, Incubation, Immunohistochemistry

Journal: Neurobiology of disease

Article Title: Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

doi: 10.1016/j.nbd.2014.05.002

Figure Lengend Snippet: Fig. 2. MBL interacts and co-localizes with JRFL gp120 in perinuclear vesicles. Confocal laser scan microscopy analysis of subcellular localization of WT MBL-EGFP (A) or Mut CS2 MBL-EGFP (B) with JRFL gp120. SK-N-SH cells for gp120 (magenta) followed by corresponding secondary antibodies and MBL WT and CS2 constructs were visualized by fluorescence of GFP (green). WT MBL-EGFP (closed arrows), (A) but not Mut CS2 MBL-EGFP (open arrows) (B) co-localized with JRFL gp120 at perinuclear vesicles. White indicates co-localization. DAPI (blue) stained the nuclei. Scale bar, 10 μm. C, CRD-dependent subcellular interaction of MBL with gp120. SK-N-SH cells were co-transfected with WT MBL-EGFP or CS2 MBL-EGFP and JRFL gp120 plas- mids with Mirus Trans IT-LT1. 36 h post-transfection cells were solubilized with 1% NP40 lysis buffer in the presence of Ca2+ and immunoprecipitated with anti-GFP polyclonal antibody. The immunoblots were probed with antibodies against gp120 and GFP respectively.

Article Snippet: Goat polyclonal antibodies specific for gp120 were obtained from Santa-Cruz Inc. (Dallas, TX) and Fitzgerald Industries International (Acton, MA).

Techniques: Microscopy, Construct, Staining, Transfection, Lysis, Immunoprecipitation, Western Blot

Journal: Neurobiology of disease

Article Title: Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

doi: 10.1016/j.nbd.2014.05.002

Figure Lengend Snippet: Fig. 4. Functional co-localization of MBL:gp120 complexes with COPII transport vesicles markers. A,C,E,G Functional co-localization of WT MBL EGFP (green) and JRFL gp120 (magenta) with ER marker (red), Golgi marker (red) and COPII transport vesicles markers (red): Sec 31A and Sec 13 (closed arrows, white indicates co-localization of three markers) in SK-N-SH cells. B, Localization of CS2 MBL (green) and JRFL gp120 (magenta) in ER (red) (open arrows). D, F, H Localization of CS2 MBL EGFP (green) and JRFL gp120 (magenta) with COP II transport vesicles: Sec 31A or Sec 13 (red) (open arrows). DAPI (blue) stained nuclei. Scale bar, 10 μm.

Article Snippet: Goat polyclonal antibodies specific for gp120 were obtained from Santa-Cruz Inc. (Dallas, TX) and Fitzgerald Industries International (Acton, MA).

Techniques: Functional Assay, Marker, Staining

Journal: Neurobiology of disease

Article Title: Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

doi: 10.1016/j.nbd.2014.05.002

Figure Lengend Snippet: Fig. 6. IIIB gp120 co-localizes with MBL and MAP2 and is transported along the microtubules in human primary neurons. Human primary neurons were untreated (A) or treated with IIIB gp120 (5 nM) for 30 min (B), 3 h (C) and 6 h (D). Neurons were fixed and stained for MBL (red), MAP2 (green), gp120 (magenta) and DAPI for nuclei (blue). Localization of MBL:MAP2 (closed arrows; yellow indicates co-localization) and MBL:MAP2:gp120 complexes (open arrows; white indicates co-localization) in neuronal soma and neurites. Dashed open arrows indicate fragmentation of the microtubules (D). Scale bar, 10 μm.

Article Snippet: Goat polyclonal antibodies specific for gp120 were obtained from Santa-Cruz Inc. (Dallas, TX) and Fitzgerald Industries International (Acton, MA).

Techniques: Staining

Journal: Neurobiology of disease

Article Title: Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

doi: 10.1016/j.nbd.2014.05.002

Figure Lengend Snippet: Fig. 7. Nocodazole blocks MBL:gp120 traffic in human primary neurons. Human primary neurons were untreated (A) or treated with nocodazole (5 μg/ml) (B) or nocodazole plus IIIB gp120 (5 nM) for 6 h. Nocodazole was added 15 min before IIIB gp120 was added. Neurons were fixed, and stained for MBL (green), MAP2 (red), gp120 (magenta) and DAPI (blue). A,B, Localization of MBL:MAP2 complexes (closed arrows; yellow indi- cates co-localization) in neurites and neuronal soma. C, Localization of MBL:MAP2: gp120 complexes predominantly in neuronal soma only (open arrows; white indicates co-localization). Scale bar, 10 μm.

Article Snippet: Goat polyclonal antibodies specific for gp120 were obtained from Santa-Cruz Inc. (Dallas, TX) and Fitzgerald Industries International (Acton, MA).

Techniques: Staining

Journal: Neurobiology of disease

Article Title: Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

doi: 10.1016/j.nbd.2014.05.002

Figure Lengend Snippet: Fig. 8. Silencing of MBL expression impaired gp120 trafficking along the neurites. SK-N-SH cells treated with shRNA control (ctrl) (A) or shRNA MBL (B) in the presence of IIIB gp120 for 30 min, were fixed and analyzed with laser scanning confocal microscopy. A, Localiza- tion and presence of MBL (red):MAP2 (green):gp120 (magenta) complexes in soma and processes of SK-N-SH cells (closed arrows; white indicates co-localization). B, Loss of MBL:MAP2:gp120 complex formation (open arrows). DAPI (blue) stains nuclei. Scale bar, 10 μm. C, Percentage of MBL remained in SK-N-SH cells based on the western blot analyses after MBL2 silencing.

Article Snippet: Goat polyclonal antibodies specific for gp120 were obtained from Santa-Cruz Inc. (Dallas, TX) and Fitzgerald Industries International (Acton, MA).

Techniques: Expressing, shRNA, Control, Confocal Microscopy, Western Blot

Journal: Neurobiology of disease

Article Title: Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

doi: 10.1016/j.nbd.2014.05.002

Figure Lengend Snippet: Fig. 9. Time lapse live cell imaging shows MBL:gp120 trafficking in subcellular dynamic vesicles. A, Time lapse microscopy of differentiated SH-SY5Y cells transfected with WT MBL-EGFP (green) and treated with 5 nM IIIB gp120 for 30 min. Time lapse was acquired as one frame every 3 s for 10 min. Arrowhead indicates anterograde (away from neuronal soma) (T = 1 to T = 100) and retrograde (towards neuronal soma) vesicle movement (T = 100 to T = 200). B, Automated vesicle tracking of a specific neurite area indicating vesicle velocities as μm/s on y-axis and relative time (seconds) on x-axis. C, Kymograph analyses of vesicle movement in the specific neurite area indicating anterograde and retrograde movement of a vesicle expressing WT MBL-EGFP and IIIB gp120. D, Co-localization of WT MBL- EGFP (green) with MAP2 (red) and gp120 (magenta) in SH-SY5Y cells (closed arrow; white indicates co-localization). DAPI (blue) stains nuclei. Scale bar, 10 μm.

Article Snippet: Goat polyclonal antibodies specific for gp120 were obtained from Santa-Cruz Inc. (Dallas, TX) and Fitzgerald Industries International (Acton, MA).

Techniques: Live Cell Imaging, Time-lapse Microscopy, Transfection, Expressing

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: Inhibition of katanin function interferes with glutamate-induced structural spine remodeling. A , Glutamate uncaging at single synapses using cultured hippocampal neurons that express either GFP or GFP-p60DEID. Left: before stimulation (black), right: after stimulation (green). Red dot: time point of uncaging stimulus. B , Quantification shows a significant difference in spine size (Δ spine size) between GFP and GFP-p60DEID after stimulation. two-way ANOVA test, significant interaction between time and condition, p < 0.0001, multiple comparison with Fisher's LSD test shows significant effects “after 8 min” p = 0.0401; “after 17 min” p = 0.0132; “after 18 min” p = 0.0393 (GFP, n = 39 spines from 14 cells; GFP-p60DEID n = 45 spines from 16 cells), 2 min bins. Before uncaging 10–0 min (black axis labels), after uncaging 0–20 min (green axis labels), five independent experiments. Individual p -values of B : before 10: p = 0.2037, before 9: p = 0.3285, before 8: p = 0.643, before 7: p = 0.4352, before 6: p = 0.4912, before 5: p = 0.8337, before 4: p = 0.4603, before 3: p = 0.9628, before 2: p = 0.1799, before 1: p = 0.2105, after 1: p = 0.8589, after 2: p = 0.1784, after 3: p = 0.0958, after 4: p = 0.1491, after 5: p = 0.1095, after 6: p = 0.0886, after 7: p = 0.1137, after 8: p = 0.0401, after 9: p = 0.0694, after 10: p = 0.0904, after 11: p = 0.1002, after 12: p = 0.4043, after 13: p = 0.3584, after 14: p = 0.0993, after 15: p = 0.0769, after 16: p = 0.1022, after 17: p = 0.0132, after 18: p = 0.0393, after 19: p = 0.0591, after 20: p = 0.3665.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Inhibition, Cell Culture, Comparison

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: Katanin inhibition affects MT growth in dendrites of premature but not mature neurons. A , Immunostaining of endogenous p60 katanin in dendrites of cultured hippocampal neurons at DIV 13–17. n = 3 experiments. B , Anti-p60 katanin immunoelectron microscopy with diaminobenzidine (DAB) showing MTs from hippocampal neurons. C,D , Anti-GFP immunoelectron microscopy with diaminobenzidine (DAB) showing MTs from hippocampal neurons transfected with either GFP-p60 katanin ( C ) or GFP-p60DEID ( D ). E , Overview image of GFP-p60 katanin (green) and endogenous p60 katanin (red). F , Magnifications of boxed region in E . G , Quantification of GFP-p60 katanin/p60 (endogenous) katanin colocalization levels. Mean ± S.E.M. = 91.16 ± 1.006%. Three independent experiments. H,I , EB3-Tomato time-lapse imaging in dendrites of DIV4 hippocampal neurons. Right: diagrams of EB3 tracks for GFP in H and for GFP-p60DEID in I . Neurons express EB3-Tomato (red) with either GFP or GFP-p60DEID (green). The green channel is not shown. J , Quantification of EB3 growth velocity (µm/s). GFP: 0.24 ± 0.01, GFP-p60DEID: 0.21 ± 0.01. Mann–Whitney U test p = 0.0152. GFP: n = 48 comets; GFP-p60DEID: n = 24 comets, three independent experiments. K,L , EB3-Tomato time-lapse imaging in dendrites of DIV14 hippocampal neurons. Right: diagrams of EB3 tracks for GFP in K and for GFP-p60DEID in L . Neurons express EB3-Tomato (red) with either GFP or GFP-p60DEID (green). The green channel is not shown. M , Quantification of K and L shows comparable growth velocity among conditions. GFP: 0.2054 + 0.01584 μm/s, GFP-p60DEID: 0.2387 + 0.01736 μm/s, Unpaired t test, two-tailed, p = 0.1712, (GFP: n = 12 ROIs, 503 comets); (GFP-p60DEID: n = 12 ROIs, 340 comets), four independent experiments.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Inhibition, Immunostaining, Cell Culture, Immuno-Electron Microscopy, Transfection, Imaging, MANN-WHITNEY, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: Functional inhibition of katanin reduces microtubule polymerization into dendritic spines. A , Immunostaining of endogenous EB3 (blue) following expression of td-Tomato (volume marker, red) together with either GFP or GFP-p60DEID (green channel not shown), DIV14 neurons. B , Quantification of % of EB3-positive spines with heads. GFP: 8.81 ± 1.35%, GFP-p60DEID: 4.90 ± 1.53%, Mann–Whitney U test p = 0.0345 (GFP: n = 33 cells, 979 spines; GFP-p60DEID: n = 20 cells, 402 spines). C , Quantification of percentage of EB3-positive stubby spines. GFP: 8.18 ± 1.50%, GFP-p60DEID: 4.86 ± 1.87%, Mann–Whitney U test, p = 0.1059 (GFP: n = 33 cells, 505 spines; GFP-p60DEID: n = 20 cells, 187 spines). D , Quantification of percentage of EB3-positive filopodia. GFP: 9.52 ± 2.90%, GFP-p60DEID: 15.26 ± 7.43%, Mann–Whitney U test, p = 0.9332 (GFP: n = 33 cells, 137 spines; GFP-p60DEID: n = 20 cells, 59 spines), three independent experiments. E , Time-lapse microscopy to investigate EB3 invasion into spines (compare with movie 7-1). Please note that the signal intensity of this example is shown with increased brightness to visualize the transient invasion and removal of the red EB3 signal (boxed region). F–H , Quantification of time-lapse video microscopy using EB3-Tomato. Please note the imaging period was 10 min, four independent experiments. F , Percentage of spines with heads invaded by EB3-Tomato over 10 min: GFP: 15.16 ± 4.43%, GFP-p60DEID: 4.54 ± 2.01%, Mann–Whitney U test, p = 0.0487 (GFP: n = 12; GFP-p60DEID: n = 13 cells). G , Percentage of stubby spines invaded by EB3-Tomato over 10 min: GFP: 12.90 ± 5.29%, GFP-p60DEID: 11.93 ± 4.51%, Mann–Whitney U test, p = 0.9794 (GFP: n = 12; GFP-p60DEID: n = 13 cells). H , Percentage of filopodia invaded by EB3-Tomato over 10 min: GFP: 22.07 ± 3.58%, GFP-p60DEID: 27.78 ± 5.82%, Unpaired two-tailed t test p = 0.4211 (GFP: n = 12; GFP-p60DEID: n = 13 cells), four independent experiments.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Functional Assay, Inhibition, Immunostaining, Expressing, Marker, MANN-WHITNEY, Time-lapse Microscopy, Microscopy, Imaging, Two Tailed Test

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: p60 katanin is located at glutamatergic spine synapses. A,B , Immunostaining of endogenous p60 katanin (green), Rhodamine phalloidin labeling of F-actin (red), DIV13–17 neurons, three independent experiments. C , Magnifications of spines from boxed regions in B . D , Fluorescence intensity profiles of line scans in C . E , Pearson's correlation coefficient indicating colocalization between p60 katanin and F-actin. Mean ± S.E.M = 0.4797 ± 0.02536, n = 18 images. F,G , Triple-immunostaining, endogenous p60 katanin (green), AMPAR subunit GluA2 (red), presynaptic marker synaptophysin (Syn, blue), DIV16–17 neurons, three independent experiments. H , Magnification of boxed regions in G . I , Fluorescence intensity profiles of line scans in H . J , Pearson's correlation coefficient between p60 and GluA2, p60 and Syn, or GluA2 (red) and GluA2 (green). p60/GluA2: mean ± S.E.M = 0.4362 ± 0.01646, n = 24 ROIs; p60/Syn: mean ± S.E.M = 0.3144 ± 0.009337, n = 24 ROIs; GluA2/GluA2: mean ± S.E.M = 0.8273 ± 0.01246, n = 30 ROIs. Kruskal–Wallis test: p60/GluA2 versus p60/Syn p = 0.0025; Kruskal–Wallis test: p60/GluA2 versus GluA2/GluA2 p < 0.0001, three independent experiments. K , Immunostaining of AMPA receptor GluA2 subunits with two fluorophores (red and green) as a positive control for the Pearson's correlation coefficient shown in Figure 2 J . L,M , Triple immunostaining of p60 katanin (red), p80 katanin (green) and PSD-95 (blue). The boxed region magnified below depicts triple colocalized puncta.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Immunostaining, Labeling, Fluorescence, Triple Immunostaining, Marker, Positive Control

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: p60 and p80 katanin are located at excitatory spine synapses. A , Immunostaining of endogenous katanin p80 (green) and Rhodamine phalloidin labeling of F-actin (red), DIV13 neurons, three independent experiments. B , Pearson's correlation coefficient indicating colocalization of p80 katanin and F-actin. Mean ± S.E.M = 0.5149 ± 0.01379, n = 25 ROIs. C , Magnifications of spines from boxed regions in A . D , Fluorescence intensity profiles of line scans in C . E , Co-immunostaining of endogenous p80 katanin (green) and AMPAR subunit GluA2 (red), DIV13–16 neurons, three independent experiments. F , Magnification of boxed regions in E . G , Fluorescence intensity profiles of line scans in F . H , Western blot analysis of p60 katanin in Triton-X-100-soluble (TS) and Triton-X-100-insoluble (TI) synaptosomal fraction. The TI fraction is also known as PSD fraction, which is enriched for postsynaptic markers (PSD-95, GluA1), but contains very little presynaptic marker (SNAP-25). n = 3 experiments. I , Anti-p60 katanin immunoelectron microscopy with diaminobenzidine (DAB) showing a katanin-negative (left) next to a katanin-positive (right, red arrow) spine synapse from hippocampal neurons.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Immunostaining, Labeling, Fluorescence, Western Blot, Marker, Immuno-Electron Microscopy

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: Analysis of GluA2 and PSD-95 signal intensities following overexpression of GFP-p60DEID. A,B , Hippocampal neurons depicting endogenous GluA2 (red), expressing either GFP (left), GFP-p60 katanin (middle) or GFP-p60DEID katanin (right), respectively. Scale bars: 15 µm (upper overview images) and 5 µm (magnifications). C , Quantification of B , the signal intensity of GluA2 in 30 µm sections of secondary dendrites of hippocampal cultures transfected either with GFP, GFP-p60 katanin, or GFP-p60DEID katanin. GFP: mean ± S.E.M = 7.199 ± 0.628, n = 24 ROIs; GFP-p60 katanin: mean ± S.E.M = 7.109 ± 0.604, n = 24 ROIs; GFP-p60DEID: mean ± S.E.M = 5.490 ± 0.461, n = 24 ROIs, three independent experiments. Kruskal–Wallis test followed by Dunn's multiple comparison test: GFP versus GFP-p60 katanin p > 0.9999; GFP versus GFP-p60DEID p = 0.1604; GFP-p60 katanin versus GFP-p60DEID p = 0.1579. D,E , Hippocampal neurons depicting endogenous PSD-95 (red), expressing either GFP (left), GFP-p60 katanin (middle), or GFP-p60DEID katanin (right), respectively. Scale bars: 15 µm (upper overview images) and 5 µm (magnifications). F , Quantification of E , the signal intensity of PSD-95 in 30 µm sections of secondary dendrites of hippocampal cultures transfected either with GFP, GFP-p60 katanin, or GFP-p60DEID katanin. GFP: mean ± S.E.M = 0.443 ± 0.054, n = 24 ROIs; GFP-p60 kat: mean ± S.E.M = 0.355 ± 0.038, n = 24 ROIs; GFP-p60DEID: mean ± S.E.M = 0.227 ± 0.029, n = 24 ROIs, three independent experiments. Ordinary one-way ANOVA test followed by Sidak's multiple comparison test: GFP versus GFP-p60 katanin p = 0.3603; GFP versus GFP-p60DEID p = 0.0014; GFP-p60 katanin versus GFP-p60DEID p = 0.0968.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Over Expression, Expressing, Transfection, Comparison

Journal: The Journal of Neuroscience

Article Title: Functional Inhibition of Katanin Affects Synaptic Plasticity

doi: 10.1523/JNEUROSCI.0374-23.2023

Figure Lengend Snippet: Functional inhibition of katanin alters synaptic transmission. A , Katanin GFP-p60DEID overexpression in primary hippocampal neurons does not alter mEPSCs under basal conditions, GFP: n = 21 neurons, GFP-p60DEID: n = 12 neurons, three independent experiments. B , Mean amplitudes (GFP: 15.03 ± 1.26 pA; GFP-p60DEID: 14.57 ± 1.21 pA, Mann–Whitney U test p = 0.9853. C , Medians of inter-event intervals (IEI). GFP: 1,952 ± 565.2 ms; GFP-p60DEID: 1,780 ± 736.3 ms, Mann–Whitney U test p = 0.5181. D , Katanin p60DEID overexpression in primary hippocampal neurons suppresses synaptic potentiation (cLTP). three independent experiments. E , EPSC amplitudes (pA). Stimulation increases the amplitudes of control transfected cells. GFP control, directly after cLTP induction: 128%; GFP control, 30 min after cLTP induction: 139%; GFP-p60DEID, directly after cLTP induction: 108%; GFP-p60DEID, 30 min after cLTP induction: 99%. 2-way ANOVA: time × condition interaction p = 0.0088. Individual data points t tests: 10 min: p = 0.0478; 30 min: p = 0.0464. 5 min bins. F , Inter-event intervals (IEIs). two-way ANOVA: time × condition no interaction p = 0.1925. Bins: 5 min. Red bar: time point of cLTP stimulus.

Article Snippet: The following antibodies were used for immunofluorescence: P60 katanin, rabbit, 1:200 (McNally F.J.), P80 katanin, rabbit, 1:200 (Proteintech), PSD-95, guinea pig, 1:200 (Synaptic Systems), P80 katanin, mouse, 1:100 (Novus), GluA2, mouse, 1:200 (Millipore), Synaptophysin, guinea pig, 1:1,000 (Synaptic Systems), EB3, rat, 1:300 (Abcam), PSD-95, mouse, 1:300 (Invitrogen), Anti-rabbit Alexa Fluor 488, donkey (Jackson), Anti-mouse Cy3, donkey (Jackson), Anti-guinea pig Cy5, donkey (Jackson), and Anti-rat Cy5, donkey (Jackson).

Techniques: Functional Assay, Inhibition, Transmission Assay, Over Expression, MANN-WHITNEY, Control, Transfection

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: CC2D1B Is Required to Maintain NE Integrity and Functionality (A) Schematic representation of CC2D1B and CHMP7. (B) GST pull-down experiments of 293T cells transiently co-expressing GST-CC2D1B together with HA-tagged CHMP2A, CHMP3, CHMP4B, or CHMP7. (C) GST pull-down experiments of 293T cells transiently co-expressing GST-CC2D1B together with HA-tagged CHMP7 full-length, CHMP7 Nt (residues 1–238), or CHMP7 Ct (residues 238–453). (D–F) Time-lapse analysis of HCT116 cells stably co-expressing GFP-NLS and H2B-mCherry and transfected with control, CHMP7 or CC2D1B siRNAs. As the recovery of nuclear integrity after cell division was previously described to be dependent on CHMP7 expression ( Olmos et al., 2016 ), CHMP7 silencing was used to validate our experiments. (D) Representation of the nucleo-cytoplasmic GFP-NLS ratio over time. Mean ± SEM; siControl n = 29; siCC2D1B n = 35, p = 0.0003; siCHMP7 n = 22, p = 0.0402. Significance compared with the control was calculated at 90 min using a two-tailed unpaired t test. (E) Representative WB of total cell lysates corresponding to siCC2D1B or siCHMP7 treated cells. (F) Representative frames corresponding to time-lapse images of siControl (top panels) or siCC2D1B (bottom panels) treated cells. Scale bars, 10 μm. (G and H) Analysis of nuclear morphology in siRNA transfected HCT116 cells. (G) Percentage of cells showing an aberrant nuclear morphology. Mean ± SEM; siControl n = 2371; siCC2D1B n = 2040, p = 0.0102; siCHMP7 n = 1887, p = 0.0024. Significance of NE invaginations compared to the control was calculated using a two-tailed unpaired t test. (H) Representative images corresponding to the quantifications shown in (G). Scale bars, 10 μm. See also Figure S1 and .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Expressing, Stable Transfection, Transfection, Control, Two Tailed Test

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: CC2D1B Is Recruited to the Reforming NE in a CHMP7-Dependent Way (A–C) Characterization of HCT116 δCC2D1B cells. (A) Total cell lysates corresponding to HCT116 Control, HCT116 δCC2D1B , HCT116 δCC2D1B /GFP and HCT116 δCC2D1B /GFP-CC2D1B were analyzed by WB using an anti-CC2D1B antibody. (B) Analysis of nuclear morphology in HCT116 δCC2D1B cells. Mean ± SEM; HCT116 Control n = 628; HCT116 δCC2D1B n = 608, p = 0.0162; HCT116 δCC2D1B /GFP n = 593, p = 0.0553; HCT116 δCC2D1B /GFP-CC2D1B n = 603, p = 0.9058. Significance of NE invaginations compared to the control was calculated using a two-tailed unpaired t test. (C) Representative images corresponding to the quantifications shown in (B). Scale bars, 10 μm. (D–F) Time-lapse images of HCT116 δCC2D1B /GFP-CC2D1B cells transfected with control (D) or CHMP7 siRNAs (E). Arrowheads indicate examples of GFP-CC2D1B perinuclear accumulation. Scale bars, 10 μm. (F) Representative WB corresponding to the cells shown in (D) and (E). (G) Time-lapse images of HCT116 δCC2D1B /GFP-CC2D1B cells stably co-expressing a mCherry-Lap2B fusion. Scale bar, 10 μm. (H) Time-lapse images of HCT116 δCC2D1B /GFP-CC2D1B cells stably co-expressing a mCherry-tubulin fusion. Scale bar, 10 μm. See also , , and .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Control, Two Tailed Test, Transfection, Stable Transfection, Expressing

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: A Functional C2 Domain Is Required for Proper Localization and Functionality of CC2D1B (A) Schematic representation of Lgd. The arrow marks the first residue of the solved structure. (B) Ribbon representation of Lgd C-terminal residues 575–816. The crystal structure was solved from a selenomethionine substituted crystal using the single wavelength anomalous dispersion method and the model refined to a resolution of 2.4 Å included the predicted C2 domain and the preceding region that lacks obvious sequence homology with known functional domains (residues 550–816). The helical domain composed of the N- and C-terminal region is shown in yellow and the C2 domain in orange. Conserved basic residues implicated in PIns(4,5)P2 interaction are shown as sticks. The N-terminal 24 residues connecting to the DM14 4 domain and present in the crystallized construct are flexible and disordered in the structure. Arrows indicate the site of insertion of the helical domain into the C2 domain. (C) Topology diagram of Lgd (residues 575–816) highlighting the insertion of the C2 domain into the helical domain. (D) Homology model of CC2D1B based on Lgd structure and docking of PIns(1,4,5)P 3 . The basic residues involved in PIns binding are shown as sticks. (E) Total cell lysates corresponding to HCT116 Control, HCT116 δCC2D1B , HCT116 δCC2D1B /GFP-CC2D1B Wt, and HCT116 δCC2D1B /GFP-CC2D1B C2M were analyzed by WB using an anti-CC2D1B antibody. (F) Time-lapse analysis of HCT116 δCC2D1B /GFP-CC2D1B Wt (top panels) or C2M (bottom panels) cells. Scale bars, 10 μm. (G) Nuclear inset of an HCT116 cell stably expressing CHMP4B-L-GFP and stained with anti-GFP, -PIP2 and -Lamin B1 antibodies. (H and I) Functional analysis of HCT116 δCC2D1B /GFP-CC2D1B C2M cells. (H) Representation of the nucleo-cytoplasmic ratio of mCherry-NLS over time in HCT116 δCC2D1B cells stably co-expressing GFP-CC2D1B Wt or C2M. Cells were incubated with Hoechst 33258 30 min before imaging to stain DNA. Mean ± SEM; HCT116 Control n = 14; HCT116 δCC2D1B n = 25, p < 0.0001; HCT116 δCC2D1B /GFP-CC2D1B Wt n = 18, p = 0.0597; HCT116 δCC2D1B /GFP-CC2D1B C2M n = 20, p < 0.0001. Significance compared to the control was calculated at 90 min using a two-tailed unpaired t test. (I) Analysis of nuclear morphology of HCT116 δCC2D1B cells stably expressing GFP-CC2D1B Wt or C2M. Mean ± SEM; HCT116 Control n = 1,055; HCT116 δCC2D1B n = 911, p = 0.0025; HCT116 δCC2D1B /GFP-CC2D1B Wt n = 896, p = 0.0651; HCT116 δCC2D1B /GFP-CC2D1B C2M n = 976, p = 0.0030. Significance of NE invaginations compared to the control was calculated using a two-tailed unpaired t test. See also Figure S2 , , and .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Functional Assay, Residue, Dispersion, Sequencing, Construct, Binding Assay, Control, Stable Transfection, Expressing, Staining, Incubation, Imaging, Two Tailed Test

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: CC2D1B Organizes the Timely Recruitment of ESCRT-III Proteins to the Reforming NE (A–C) Time-lapse analysis of HCT116 cells stably expressing GFP-CHMP7 (A), or stably co-expressing CHMP4B-L-GFP (B) or CHMP2A-L-GFP (C) along with H2B-mCherry. Cells were transfected with control or CC2D1B siRNAs. Scale bars, 10 μm in (A), (B), and (C). (D) Quantification of GFP-CHMP7, CHMP4B-L-GFP, and CHMP2A-L-GFP recruitment to the reforming NE in HCT116 cells transfected with control (top panel) or CC2D1B (bottom panel) siRNAs. T0 was set at the beginning of furrow ingression (FI). Curves indicate mean ± SEM. GFP-CHMP7: siControl n = 7; siCC2D1B n = 7. CHMP4B-L-GFP: siControl n = 11; siCC2D1B n = 13. CHMP2A-L-GFP: siControl n = 15; siCC2D1B n = 15. (E and F) Recruitment of endogenous CHMP2A to the reforming NE in HCT116 fixed cells. (E) Representative images of HCT116 cells transfected with control or CC2D1B siRNAs and stained with anti-CHMP2A and anti-tubulin antibodies. (F) Quantification of the midzone distance in CHMP2A-positive cells. Mean ± SEM; siControl n = 29; siCC2D1B n = 32, p = 0.0004. Scale bars, 10 μm in (E). (G) Sucrose gradient analysis of MBP-CHMP4B ΔC-Alix polymerization in the presence of CC2D1B (residues 317–558). Upper panel, CC2D1B (residues 317–558) floats in the top fractions of the gradient consistent with being monomeric; second panel, MBP-CHMP4B ΔC-Alix is found in the top fractions (monomers) and in the bottom (polymers) fraction; third panel, TEV cleavage of monomeric MBP-CHMP4B ΔC-Alix induces polymerization as indicated by the band in the bottom fraction; lower panel, TEV cleavage of monomeric MBP-CHMP4B ΔC-Alix in the presence of CC2D1B (residues 317–558) retains CHMP4B ΔC-Alix in the monomer fractions. (H) Comparison of CC2D1B and CHMP4B recruitment times. HCT116 cells expressing GFP-CC2D1B or CHMP4B-L-GFP were analyzed by time-lapse microscopy and the first (Ti) and the last (Tf) frames showing GFP-CC2D1B or CHMP4B-L-GFP accumulation in the perinuclear area were scored. Whiskers mark 5–95 percentiles. The movies used to quantify CHMP4B-L-GFP Ti and Tf were the same used for the recruitment quantification shown in Figure 4 D. T0 was set at the beginning of furrow ingression (FI). GFP-CC2D1B n = 29; Ti p = 0.0183; Tf p < 0.0001. Significance compared to the control was calculated using a two-tailed unpaired t test. (I) Analysis of GFP-CC2D1B C2M expression on ESCRT-III recruitment time. HCT116 cells co-expressing CHMP2A-L-mCherry and GFP-CC2D1B Wt or C2M were analyzed by time-lapse microscopy and the first (Ti) and the last (Tf) frames showing CHMP2A-L-mCherry accumulation in the perinuclear area were scored. Whiskers mark 5–95 percentiles. T0 was set at the beginning of furrow ingression (FI). GFP-CC2D1B Wt n = 10; GFP-CC2D1B C2M n = 13; Ti p = 0.0803; Tf p = 0.0638. Significance compared to the control was calculated using a two-tailed unpaired t test. See also Figure S4 and Video S8. The Silencing of CC2D1B Induces the Premature Recruitment of GFP-CHMP7 to the Resealing NE, Related to Figure 4A , Video S9. The Silencing of CC2D1B Induces the Premature Recruitment of CHMP4B-L-GFP to the Resealing NE, Related to Figure 4B , Video S10. The Silencing of CC2D1B Induces the Premature Recruitment of CHMP2A-L-GFP to the Resealing NE, Related to Figure 4C , , .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Stable Transfection, Expressing, Transfection, Control, Staining, Comparison, Time-lapse Microscopy, Two Tailed Test

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: CC2D1B Coordinates NM Deposition with ESCRT-III Recruitment to the Resealing NE (A) Time-lapse analysis of HeLa cells stably co-expressing YFP-Sec61β and H2B-mCherry. Cells were transfected with control or CC2D1B siRNAs. Scale bars, 10 μm. (B) Time-lapse analysis of HeLa cells stably co-expressing CHMP4B-L-GFP and H2B-mCherry. Cells were transfected with control or CC2D1B siRNAs. Scale bars, 10 μm. (C) Quantification of YFP-Sec61β (Top panel. Lines: mean ± SEM) or CHMP4B-L-GFP (Bottom panel. Curves: mean ± SEM) recruitment to the NE in HeLa cells transfected with control or CC2D1B siRNAs. T0 was set at the beginning of furrow ingression (FI). YFP-Sec61β recruitment: siControl n = 15; siCC2D1B n = 10. CHMP4B-L-GFP recruitment: siControl n = 11; siCC2D1B n = 14. (D) Super resolution time-lapse analysis of HCT116 cells stably co-expressing CHMP4B-L-GFP and mCherry-Emerin and transfected with control or CC2D1B siRNAs. The frames corresponding to the beginning of CHMP4B recruitment (CHMP4B Ti) to the reforming NE are shown. (E) Quantification of the percentage of NE reformation at the beginning of CHMP4B recruitment (CHMP4B Ti). Bars indicate median. siControl n = 9; siCC2D1B n = 6, p = 0.0027. Significance compared to the control was calculated using a two-tailed unpaired t test. See also Figure S5 and .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Stable Transfection, Expressing, Transfection, Control, Two Tailed Test

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: CC2D1B Silencing Impairs Spastin Activity (A) Time-lapse analysis of HCT116 cells stably co-expressing GFP-Spastin M87 and H2B-mCherry. Cells were transfected with control, CC2D1B, or CHMP7 siRNAs. Scale bars, 10 μm. (B) GFP-Spastin M87 fluorescence recruitment to the reforming NE in HCT116 cells transfected with control or CC2D1B siRNAs. T0 was set at the beginning of furrow ingression (FI). Curves indicate mean ± SEM. siControl n = 18; siCC2D1B n = 30. (C) Representative WB corresponding to the cells shown in (A). (D) Area under the curves corresponding to the recruitment of GFP-Spastin M87 fluorescence to the reforming NE shown in Figure 6 B. Cells were transfected with control or CC2D1B siRNAs. Bar indicates median. p = 0.0008. Significance compared to the control was calculated using a two-tailed unpaired t test. (E) Time-lapse analysis of HCT116 cells stably co-expressing mCherry-tubulin with CHMP2A-L-GFP and transfected with control, Spastin, or CC2D1B siRNAs. Scale bars, 10 μm. (F–I) Quantification of nuclear or cytoplasmic tubulin fluorescence over time in HCT116 cells stably co-expressing mCherry-tubulin with CHMP2A-L-GFP and transfected with Spastin (F and G) or CC2D1B (H and I) siRNAs. T0 was set at the beginning of furrow ingression. siControl n = 10; siCC2D1B n = 8; siSpastin n = 9. (F and H) Nuclear tubulin fluorescence (lines indicate mean ± SEM) siSpastin p = 0.0158; siCC2D1B p = 0.0294. CHMP2A-L-GFP recruitment to the NE (curve indicates mean ± SEM) in control cells. (G and I) Cytoplasmic tubulin fluorescence (lines indicate mean ± SEM) siSpastin p = 0.0099; siCC2D1B p = 0.0007. Significance compared to the control was calculated at 600 s using a two-tailed unpaired t test. See also Figure S6 and .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Control, Fluorescence, Two Tailed Test

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet: Persistent Transnuclear Microtubules Originate Deep Nuclear Invaginations in Interphase (A) z stack series corresponding to an HCT116 cell stably co-expressing mCherry-tubulin with CHMP2A-L-GFP, transfected with CC2D1B siRNA, and incubated with Hoechst 33258 30 min before imaging to stain DNA. The signal corresponding to Hoechst 33258 is shown in green. The signal corresponding to CHMP2A-L-GFP is not shown. This cell was selected as an example of a nucleus showing transnuclear microtubule tunnels. Scale bar 10 μm. (B) Frequency of transnuclear microtubule tunnels in HCT116 cells stably co-expressing mCherry-tubulin with CHMP2A-L-GFP and transfected with control, Spastin, or CC2D1B siRNAs. Mean ± SEM; siControl n = 1,083; siCC2D1B n = 850, p = 0.0145; siSpastin n = 1,075, p = 0.0007. Significance compared to the control was calculated using a two-tailed unpaired t test. (C and D) 3D rendering of the cell shown in (A). (C) Front view corresponding to Hoechst 33258 channel. (D) Top view corresponding to Hoechst 33258 and mCherry-tubulin. 80% transparency was applied to Hoechst 33258 channel to facilitate the observation of transnuclear microtubule channels. Scale bars, 5 μm. (E) Proposed model. Left panel: (1) CC2D1B binds to the monomeric cytoplasmic form of CHMP4B, hindering its association with CHMP7 and therefore preventing its premature recruitment to the NE. (2) By means of its ability to interact with CHMP7 and CHMP4B through its N-terminal DM14 domains and with membrane phospholipids through its C-terminal C2 domain, CC2D1B could function as a scaffold to position CHMP4B monomers close to the NM. Then, CC2D1B could mediate the organization of a transient CHMP7-CC2D1B-CHMP4B complex at the sealing gaps of the NE, facilitating the delivery of CHMP4B monomers to the growing ESCRT-III filament. (3) Spastin is recruited by the ESCRT-III to catalyze the severing of spindle microtubules, which facilitates the final sealing of the NE. Right panel: (1) in CC2D1B-silenced cells, CHMP4B monomers are free to associate to CHMP7 without restrictions, (2) triggering the premature polymerization of CHMP4B monomers on the NE. (3) These prematurely formed ESCRT filaments cannot be competent for Spastin recruitment and membrane constriction, which results in an impaired ability of the cells to sever spindle microtubules, leading to deleterious effects in nuclear integrity and functionality. See also Figure S7 and .

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Stable Transfection, Expressing, Transfection, Incubation, Imaging, Staining, Control, Two Tailed Test, Membrane

Journal: Developmental Cell

Article Title: CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope

doi: 10.1016/j.devcel.2018.11.012

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were: rabbit CC2D1B (H-258, Santa Cruz Biotechnology), rabbit CHMP7 (16424-1-AP, Proteintech), mouse Spastin (Sp 3G11/1, Santa Cruz Biotechnology), mouse GFP clones 7.1 and 13.1 (11814460001, Roche), rabbit Hsp90 (H-114, Santa Cruz Biotechnology), mouse Hsp90 (F-8, Santa Cruz Biotechnology), mouse GST (B-14, Santa Cruz Biotechnology), rabbit HA (ABIN100176, Antibodies Online).

Techniques: Virus, Recombinant, Protease Inhibitor, Software

Journal: Nature Communications

Article Title: The HIV co-receptor CCR5 regulates osteoclast function

doi: 10.1038/s41467-017-02368-5

Figure Lengend Snippet: The locomotive and adhesion defects in Ccr5 -deficient ( Ccr5 − / − ) osteoclasts. a Time-lapse microscopy frames show the locomotion of wild-type ( Ccr5 + / + ) and Ccr5 − / − osteoclasts expressing GFP (scale bars, 100 μm). The patterns of cellular motility were scored with the ratios of the contraction (shown in red) and extension (in green) areas. The contraction and extension areas were scored by 10 min interval time-lapse images. Time-laps movies are shown in Supplementary Movies and . GFP-expressing multi-nucleated cells from wild-type and Ccr5 − / − mice were analyzed ( n = 9 and 6, respectively). Cells showing the parameters closest to the mean values are shown. b The ratios of the contraction and extension areas, and the cell deformation index (CDI) were analyzed and statistically compared. * P < 0.05 by Student’s t -test. The data shown as the mean ± SD ( n = 6–7). c , d BMCs obtained from wild-type and Ccr5 − / − cells were subjected to immunoblotting of phosphorylated Src, Pyk2, NF-κB, p38 and ERK, and Akt. Prior to the analyses, the cells were stimulated by RANKL for the indicated time. The immunoblotting data were replicated more than three times. e Three-dimensional (3D)-SIM images demonstrate the assembly of actin-enriched podosome cores, Pyk2, and vinculins in mature wild-type and Ccr5 − / − osteoclasts. The cells were subjected to immunohistochemical staining using anti-Pyk2 antibodies (shown in red) and anti-vinculin antibodies (shown in pink), and were concomitantly stained with phalloidin-AlexaFluor488 to visualize the actin rings (shown in green). Maximum intensity projection images of 3D-SIM optical slices are shown (scale bars, 5 μm, n = 4). Reconstructed 3D-SIM images are shown in Supplementary Movies and

Article Snippet: A Mouse RANKL ELISA kit (R&D system), Mouse Osteocalcin/Bone gla protein, OT/BGP ELISA kit (Biomedical Technologies, Stoughton, MA), M-CSF (R&D system), Osteoprotegerin/TNFRSF11B ELISA kit (R&D system).

Techniques: Time-lapse Microscopy, Expressing, Western Blot, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: The HIV co-receptor CCR5 regulates osteoclast function

doi: 10.1038/s41467-017-02368-5

Figure Lengend Snippet: CCL5 stimulates osteoclastogenesis through adhesion signaling. a Double immunofluorescence staining of the trabecular bone in the wild-type mouse tibia (4-week-old, male) with anti-Cathepsin K (shown in red) and anti-CCR5 (in green) antibodies. The nuclei were stained with DAPI. Staining of Ccr5 -deficient bone sections are also shown to confirm the specificity of anti-CCR5 antibodies. Magnification (objective lens) ×40 (left and right panels, wide-field fluorescence microscopy, scale bars, 20 μm) and ×100 (middle panel, SIM, scale bar, 20 μm). BM bone marrow, n = 3. b The relative mRNA expression levels of Ccr5 during osteoclastogenesis were measured by a real-time Q-PCR (mean ± SD, n = 4). c , d The effect of CCL5 on osteoclastogenesis was revealed by TRAP staining (scale bar, 100 μm). BMCs that were isolated from Ccr5 + / + and Ccr5 − / − mice were cultured with M-CSF and RANKL with or without recombinant CCL5. The number of multi-nucleated osteoclasts following exogenous CCL5 treatment was quantified (mean ± SD, n = 4). * P < 0.05 by Student’s t -test. e The effects of CCL5 on focal adhesion signal were investigated by immunoblotting. BMCs isolated from wild-type mice were cultured with M-CSF and RANKL for 3 days and preincubated with CCL5 for 30 min prior to sRANKL stimulation for the indicated time. The phosphorylation levels of FAK, Src, and Pyk2 were analyzed. Immunoblotting data were replicated more than three times. f BMCs were treated with RANKL alone or the combination of RANKL and CCL5 (100 ng mL −1 ). Total cell lysates immunoprecipitated with phospho-tyrosine kinase and immunoblotted with phospho-Src. g BMCs isolated from wild-type mice were cultured for 3 days with M-CSF and RANKL to induce osteoclast precursors and then treated with rmCCL5 for the indicated time after serum starvation for 30 min. Total cell lysates were harvested to analyze the activity levels of GTP-Rac and -RhoA by a G-LISA (mean ± SD, n = 5). * P < 0.05 by Student’s t -test. h – j A gene ontology analysis of the genes that showed significantly altered expression levels in RNA-sequencing using cells incubated with RANKL alone and the combination of RANKL and CCL5 (100 ng mL −1 ) (three analyses per condition). The significantly upregulated pathways ( i ) and heat maps of the obtained results ( j ) are shown. Red, high expression; green, low expression

Article Snippet: A Mouse RANKL ELISA kit (R&D system), Mouse Osteocalcin/Bone gla protein, OT/BGP ELISA kit (Biomedical Technologies, Stoughton, MA), M-CSF (R&D system), Osteoprotegerin/TNFRSF11B ELISA kit (R&D system).

Techniques: Double Immunofluorescence Staining, Staining, Fluorescence, Microscopy, Expressing, Isolation, Cell Culture, Recombinant, Western Blot, Phospho-proteomics, Immunoprecipitation, Activity Assay, RNA Sequencing, Incubation

Journal: Nature Communications

Article Title: The HIV co-receptor CCR5 regulates osteoclast function

doi: 10.1038/s41467-017-02368-5

Figure Lengend Snippet: The functional rescue of the Ccr5 − / − osteoclasts by active small GTPases. a Immunoblotting analyses of Vav3 and phosphorylated FAK were conducted 15 min after RANKL stimulation in wild-type and Ccr5 − / − BMCs. The immunoblotting data were replicated more than three times. b The levels of the active forms of Rac1, RhoA, and Cdc42 in osteoclastic differentiation were analyzed in cells from wild-type and Ccr5 − / − bones (mean ± SD, n = 5). c The adhesion ring formation of wild-type and Ccr5 − / − osteoclasts expressing the indicated constructs was examined in cells cultured on a glass-bottomed dish, and then analyzed by anti-vinculin immunofluorescence staining (shown in green, scale bars, 100 μm). Magnification ×20 (objective lens), n = 4. The vinculin intensity per cell perimeter was quantified and statistically compared (mean ± SD, n = 10). * P < 0.05 by Student’s t -test. d Resorption pit assays. After culturing, the osteoclasts were removed from the dentin slices and were subsequently stained with hematoxylin to visualize the resorption pits (scale bars, 100 μm, n = 5). The resorption pit areas (%) on images were scored and statistically compared. e The relative mRNA levels of Integrin-αV , Mmp3 , and Mmp13 were measured by a real-time Q-PCR (mean ± SD, n = 4). * P < 0.05 by Student’s t -test

Article Snippet: A Mouse RANKL ELISA kit (R&D system), Mouse Osteocalcin/Bone gla protein, OT/BGP ELISA kit (Biomedical Technologies, Stoughton, MA), M-CSF (R&D system), Osteoprotegerin/TNFRSF11B ELISA kit (R&D system).

Techniques: Functional Assay, Western Blot, Expressing, Construct, Cell Culture, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: The HIV co-receptor CCR5 regulates osteoclast function

doi: 10.1038/s41467-017-02368-5

Figure Lengend Snippet: Ccr5 -deficient male mice are resistant to RANKL-induced bone loss. a , b Micro-computed tomography (μCT) images (scale bars, 100 μm) and the analysis of the femurs of 7-week-old Ccr5 − / − and their wild-type littermates ( Ccr5 +/+ ) ( n = 4–6 mice per group). The BMD, BV/TV, Tb.N, and Conn-Dens were scored and statistically compared. c The levels of serum mouse RANKL, OPG, TRAP, and CTX were measured by an ELISA, and statistically compared. d Representative images of histological sections of the distal femurs obtained from wild-type and Ccr5 − / − mice are shown. The sections were stained to show the activity level of TRAP (shown in red), and Villanueva staining was performed to reveal the osteoid and osteoblasts (in pale purple); the nuclei were revealed by toluidine blue staining (in blue). The stained sections were observed by differential interference contrast (DIC) microscopy. Magnification (objective lens): ×10 (upper panel), ×20 (middle panel), and ×40 (lower panel), respectively. Scale bars, 100 μm, n = 4–6. e Quantitative bone histomorphometric analyses were conducted of the trabecular bones in the distal femurs of Ccr5 + / + and Ccr5 − / − mice. The osteoclast number (N.Oc), osteoclast surface per bone surface (Oc.S/BS), osteoclast number per bone perimeter (N.Oc/B.Pm) and osteoclast number per osteoclast perimeter (N.Oc./Oc.Pm.) were scored and statistically compared. Each sample was duplicated. * P < 0.05 (by one-way analysis of variance (ANOVA)) in comparison to WT or RANKL-injected WT. All values are shown as the mean ± SD, n = 4–6

Article Snippet: A Mouse RANKL ELISA kit (R&D system), Mouse Osteocalcin/Bone gla protein, OT/BGP ELISA kit (Biomedical Technologies, Stoughton, MA), M-CSF (R&D system), Osteoprotegerin/TNFRSF11B ELISA kit (R&D system).

Techniques: Micro-CT, Enzyme-linked Immunosorbent Assay, Staining, Activity Assay, Microscopy, Comparison, Injection

Journal: Nature Communications

Article Title: The HIV co-receptor CCR5 regulates osteoclast function

doi: 10.1038/s41467-017-02368-5

Figure Lengend Snippet: The blockade of CCL5 in vivo. a The serum levels of CCL3 and CCL5 in 10-week-old mice were measured by an ELISA ( n = 5). b Mouse anti-CCL5 neutralizing antibodies (mCCL5 neu ab, 500 μg per mouse) were injected (once per week for 2 weeks) into 6-week-old male C57BL/6J mice ( n = 5). IgG was administered to the control group. c , d μCT images (scale bars, 100 μm) and parameters ( n = 5 mice per group) are shown. e Representative images of the distal femurs from the control IgG and mCCL5 ab groups. HE- (scale bars, 100 μm) and TRAP-stained sections (scale bars, 50 μm) are shown in the upper and lower panels, respectively ( n = 5). f Quantitative bone histomorphometric analyses were conducted of the trabecular bones in the distal femurs of control IgG and mouse CCL5 neutralizing antibody-injected mice. * P < 0.05 (by Student’s t -test) in comparison to control and mCCL5-neuAb mice. All values are shown as the mean ± SD, n = 5

Article Snippet: A Mouse RANKL ELISA kit (R&D system), Mouse Osteocalcin/Bone gla protein, OT/BGP ELISA kit (Biomedical Technologies, Stoughton, MA), M-CSF (R&D system), Osteoprotegerin/TNFRSF11B ELISA kit (R&D system).

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Control, Staining, Comparison, Neutralizing Assay

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Galectin-3 bound to Lm332 via β-galactoside. A, purified Lm332s (1 μg), vector-Lm332 (vecLm332), GnT-III-Lm332 (III-Lm332), and GnT-V-Lm332 (V-Lm332), were run on 5–15% gradient gels under reducing conditions. After SDS-PAGE, separated proteins were stained with Coomassie Brilliant Blue. Ordinates indicate molecular sizes in kDa of marker proteins and laminin chains. Asterisk indicates the expected position of galectin-3. B, detection of galectin-3 co-purified with Lm332. 100 ng of purified Lm332s (right lanes), vector-Lm332 (vecLm332), GnT-III-Lm332 (III-Lm332), and GnT-V-Lm332 (V-Lm332), and 20 μg of cell lysate from keratinocytes (left lane) were run on a 12% SDS-polyacrylamide gel, blotted onto a nitrocellulose membrane, and then probed with an anti-galectin-3 Ab. I.B., immunoblot. C, pulldown assay of recombinant galectin-3 with Lm332. GST-Sepharose beads bound with purified GST (GST) and GST-galectin-3 (gal3) were incubated with Lm332. After incubation, the beads were thoroughly washed, and bound Lm332 was eluted using 0.2 m lactose. Bound Lm332 was confirmed by immunoblotting using an anti-laminin β3 chain mAb. Other procedures are described under “Experimental Procedures.” D, inhibitory assay of galectin-3 binding to purified Lm332. 2 μg/ml Lm332 was used to coat 96-well ELISA plates at 4 °C overnight. After blocking with 1.2% BSA, purified GST or GST-galectin-3 protein in keratinocyte supplement-free medium containing no additives, 0.2 m sucrose, 0.2 m lactose, or 10 mm EDTA was added to the wells followed by incubation for 1 h at 37 °C. The amount of galectin-3 bound to Lm332 was estimated by ELISA using an anti-GST Ab. Each bar represents the mean ± S.D. of triplicate assays. E, 96-well ELISA plates were coated with vector-Lm332 (vec), GnT-III-Lm332 (GnT-III), or GnT-V-Lm332 (GnT-V) at a concentration of 2 μg/ml at 4 °C overnight. After blocking with 1.2% BSA, the purified galectin-3 protein in keratinocyte supplement-free medium was added to the wells, followed by incubation for 1 h at 37 °C. Bound galectin-3 was estimated by ELISA using an anti-GST Ab. Each bar represents the mean ± S.D. of triplicate assays.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Purification, Plasmid Preparation, SDS Page, Staining, Marker, Western Blot, Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Galectin-3 promoted Lm332-dependent cell migration. A, detection of Lm332 expression in NIK and laminin β3 chain null keratinocytes. Cell lysates (20 μg) from the indicated keratinocytes were run on a 7.5% SDS-polyacrylamide gel, blotted onto a nitrocellulose membrane, and then probed with an anti-laminin β3 Ab and then reprobed with an anti-α-tubulin Ab. IB, immunoblot. B, effect of galectin-3 on Lm332-dependent cell migration. Lm332 null keratinocytes in culture medium containing 6.3 μg/ml GST or purified galectin-3 (gal3) protein were plated onto dishes that had been precoated with 2 μg/ml vector-Lm332 (vecLm332) or GnT-III-Lm332 (III-Lm332), followed by incubation for 1 h. Cell migration on each substrate was monitored by time-lapse microscopy as described under “Experimental Procedures.” Each bar represents the mean ± S.D. of the migration distance of eight cells in each assay in three independent experiments. C, effects of anti-galectin-3 function-blocking mAb (gal3 mAb) on cell migration on the indicated Lm332 substrates were monitored by time-lapse microscopy. Rat IgG was used as a control (IgG). D, NIK cells and Lm332 null keratinocytes were grown to confluency on collagen-coated dishes. After the cell monolayer was scratched, cells were incubated in keratinocyte growth medium for 8.5 h, and photographs were then taken. Dashed lines indicate the borders between cell and wound cell-free area at 0 h.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Migration, Expressing, Western Blot, Purification, Plasmid Preparation, Incubation, Time-lapse Microscopy, Blocking Assay

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Galectin-3 suppressed cell spreading on Lm332 during the initial stage of cell adhesion. A, effects of anti-galectin-3 Ab and lactose on cell attachment. Keratinocytes in suspension were incubated with a control rat IgG (IgG), anti-galectin-3 functional blocking mAb (gal3 mAb), sucrose, or lactose for 20 min at room temperature and then added to the vector-Lm332-precoated plates for 10 min at 37 °C. Each bar represents the mean ± S.D. of triplicate assays. B, effects of galectin-3 on cell spreading. The cell morphology was photographed after cell spreading on Lm332 for 15 min in the presence of GST or galectin-3. C, relative value of cell spreading area in each condition. Each bar represents the mean ± S.D. of 25 cells.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Cell Attachment Assay, Incubation, Functional Assay, Blocking Assay, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Effects of galectin-3 on Lm332-mediated α3β1 integrin clustering and focal contact formation. Lm332-null keratinocytes were treated with control IgG (IgG) or anti-galectin-3 functional blocking mAb (gal3 mAb) and then plated on the indicated Lm332s. After incubation for 1.5 h, cells were fixed and stained with α3 integrin (A) and paxillin (B) mAb and followed by secondary antibodies. Arrowheads indicate α3β1 integrin clustering (A) and focal contacts (B). Scale bar, 20 μm.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Functional Assay, Blocking Assay, Incubation, Staining

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Effects of N-glycosylation on Lm332-mediated α6β4 integrin clustering. A, Lm332-null keratinocytes were added to vector-Lm332- (vecLm332) or GnT-III-Lm332 (III-Lm332)-coated plates. B, cells were plated on vector-Lm332 and incubated for 1.5 h in the presence of control rat IgG (IgG) or anti-galectin-3 functional blocking mAb (gal3 mAb). Cells were fixed and then stained with an α6 integrin mAb. Arrowheads indicate α6β4 integrin clustering. Scale bar, 20 μm.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Plasmid Preparation, Incubation, Functional Assay, Blocking Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Galectin-3-mediated complex formation via β-galactoside. A, detection of galectin-3-β4 integrin-EGFR complex. Keratinocytes were subjected to DTSSP cross-linking, and then cell lysates were immunoprecipitated (IP) using the indicated antibodies or a control IgG. Immunoprecipitates were run on either a 6% (β4 and EGFR) or 12% (galectin-3) SDS-polyacrylamide gel and probed with the indicated Ab. IB, immunoblot; gal3, galectin-3. B, galectin-3-β4 integrin-EGFR complex was disrupted by lactose. Keratinocytes were cultured in keratinocyte growth medium containing 0.2 m sucrose or lactose for 24 h, and galectin-3 was then added to the cell culture medium. After incubation for 2 h at 37 °C, cells were subjected to DTSSP cross-linking. After immunoprecipitation with EGFR Ab, precipitates were blotted onto the membrane and probed with a β4 integrin Ab. Membranes were reprobed with an anti-EGFR Ab. Results of the densitometric analysis are shown as the integrated density of the ratio of β4 integrin to EGFR bands, and the ratio in NIK cells treated with sucrose was equal to 1. The same amount of each cell lysate, which was used for immunoprecipitation, was further confirmed by immunoblotting with an anti-α-tubulin Ab. C, binding of recombinant galectin-3 on the keratinocyte surface. 2.5 μg/ml purified GST (shaded histogram) or 5.0 μg/ml galectin-3 protein was added to the keratinocyte culture medium. After incubation for 24 h at 37 °C, the protein bound on the keratinocyte surface was analyzed by fluorescence-activated cell sorter using an anti-GST Ab. D, galectin-3 enhanced ERK phosphorylation. 2.5 μg/ml GST or 5.0 μg/ml galectin-3 was added to the keratinocyte culture medium, followed by incubation for 24 h. The collected lysate in each condition was probed with phospho-ERK Ab and reprobed with ERK1 Ab.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Incubation, Binding Assay, Recombinant, Purification, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Expression of GnT-III suppressed galectin-3-mediated complex formation in MKN45 transfectants. A, vector- and GnT-III-MKN45 transfectants were subjected to DTSSP cross-linking, and then the cell lysates were immunoprecipitated (IP) using anti-β4 integrin antibody to detect β4 integrin complex. Immunoprecipitates were run on either a 6% (β4 and EGFR) or 12% (galectin-3) SDS-polyacrylamide gel and probed with the indicated Abs. IB, immunoblot. B, 2.5 μg/ml GST or 5.0 μg/ml galectin-3 was added to cell cultures of the indicated MKN45 transfectants, followed by incubation for 24 h. The collected lysate in each condition was probed with phospho-ERK Ab or ERK1 Ab. Results of the densitometric analysis are shown as the integrated density of the ratio of phospho-ERK1/2 to total ERK1 bands, and the ratio in vector-MKN45 transfectants treated with GST was equal to 1.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Bisecting GlcNAc Residues on Laminin-332 Down-regulate Galectin-3-dependent Keratinocyte Motility *

doi: 10.1074/jbc.M109.038836

Figure Lengend Snippet: Model for the inhibitory effects of GnT-III on cell migration and signal transduction. Under normal conditions, galectin-3 binds to integrins, ECM, and growth factor receptors to form a complex on the cell surface for cellular signaling and cell migration in cancer cells or normal cells, such as keratinocytes, which usually express high or moderate levels of GnT-V and, therefore, contain some poly-N-acetyllactosamine N-glycans. However, overexpression of GnT-III results in modification of glycoproteins by bisecting GlcNAc, which inhibits GlcNAc branch formation catalyzed by GnT-IV and GnT-V, thereby suppressing addition of poly-N-acetyllactosamine N-glycans on these proteins. Therefore, in this case, galectin-3 cannot form the signaling platform consisting of molecules such as EGFR, Lm332, and α3β1 and α6β4 integrins, inhibiting both cellular signaling and cell migration.

Article Snippet: The following antibodies were used in this study: rat monoclonal antibodies (mAbs) specific for galectin-3 (M3/38) and α6 (GoH3); rabbit polyclonal Abs to EGFR (1005), glutathione S -transferase (GST) (z-5), and β4 integrin (H101) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse mAbs to the human laminin β3 chain (Kalinin B1), ERK1 (MK12), and paxillin (349) (BD Transduction Laboratories, Lexington, KY); and a mouse mAb to phospho-ERK1/2 (E10) from Cell Signaling Technology.

Techniques: Migration, Transduction, Over Expression, Modification

Journal: The Journal of Biological Chemistry

Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

doi: 10.1074/jbc.M113.542688

Figure Lengend Snippet: GOLPH3 was associated with sialyltransferases through the cytoplasmic domain of sialyltransferase. A, schematic diagram of sialyltransferases and chimeric constructs. GOLPH3 and the chimera of ST3GAL4 and β4GALT1 (B) or the chimera of ST6GAL1 and β4GALT1 (C) were transiently expressed in 293T cells. The cell lysates were immunoprecipitated (IP) with anti-FLAG and immunoblotted with anti-HA or anti-FLAG antibody. D, WT or GOLPH3 mutants (R171A/R174A, W81A/R90A, Δ190–201) shRNA-resistant in a Tet-inducible expression system were introduced into HeLa cells that expressed the Tet-inducible shRNA GOLPH3-292 (KD), as described under “Experimental Procedures.” Cells were treated with 1 μg/ml of doxycycline for 72 h, lysed, and immunoprecipitated with SSA-agarose and immunoblotted with anti-β1 integrin. E, to examine the effects of GOLPH3 knockdown on localization of ST6GAL1, those ST6GAL1-GFP cells expressed with the doxycycline (DOX)-inducible GOLPH3 knockdown system were cultured for 72 h in the presence (KD) or absence (Ctrl) of DOX. Cells were stained with anti-GM130 primary antibody, TO-PRO-3, and fluorescent secondary antibodies. The cells were analyzed using an Olympus fluorescence microscope with 60×/1.35 NA oil immersion objective lens (FV1000 system). Scale bar, 10 μm.

Article Snippet: The following antibodies were used: monoclonal antibodies against α5 (610634) and β1 (610468) from BD Biosciences; monoclonal antibody against α-tubulin (Sigma, T6199), β1 integrin for immunoprecipitation (TS2/16, ATCC); FACS or functional blocking (P5D2, DSHB); HA (Roche Applied Science, 1867423); antibody against FLAG for immunoblot (Sigma, F1804); immunoprecipitation (Sigma, A2220); polyclonal antibody against GOLPH3 (Abcam, ab82377); α3 integrin (Santa Cruz Biotechnology, Sc-6592); polyclonal antibody against EGFR (2232); AKT (9272); and monoclonal antibody against pAKT Ser-473 (4060) from Cell Signaling.

Techniques: Construct, Immunoprecipitation, shRNA, Expressing, Knockdown, Cell Culture, Staining, Fluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

doi: 10.1074/jbc.M113.542688

Figure Lengend Snippet: Effects of knockdown of GOLPH3 on tumorigenicity and integrin-mediated cell migration. A, expression levels of GOLPH3 in HeLa cells after transfection with GOLPH3 siRNAs (KD) were compared with cells transfected with nontargeting siRNAs (Ctrl). B, cell migration toward FN was determined using the transwell assay as described under “Experimental Procedures.” Cells that migrated were stained with crystal violet. Migrated cells were counted under a microscope. A representative example is shown in the left panel. The scale bar, 100 μm. The quantitative data (right panel) were obtained from three independent experiments. Data are presented as the means ± S.D. (**, p < 0.001 by one-way ANOVA with Tukey's post hoc test). C, xenograft model in nude mice. A total of 1 × 106 cells expressed shRNA for control (Ctrl) or GOLPH3 (KD) were inoculated on the backs of nude mice by hypodermic injection. After inoculation for 6 weeks, tumor sizes were measured as described under “Experimental Procedures.” The quantitative data for the tumor volume (left panel) and tumor weight (right panel) are presented as the means ± S.D. (n = 5; **, p < 0.001 by one-tail unpaired t test). Ctrl, control; KD, GOLPH3-knockdown; β1, integrin β1.

Article Snippet: The following antibodies were used: monoclonal antibodies against α5 (610634) and β1 (610468) from BD Biosciences; monoclonal antibody against α-tubulin (Sigma, T6199), β1 integrin for immunoprecipitation (TS2/16, ATCC); FACS or functional blocking (P5D2, DSHB); HA (Roche Applied Science, 1867423); antibody against FLAG for immunoblot (Sigma, F1804); immunoprecipitation (Sigma, A2220); polyclonal antibody against GOLPH3 (Abcam, ab82377); α3 integrin (Santa Cruz Biotechnology, Sc-6592); polyclonal antibody against EGFR (2232); AKT (9272); and monoclonal antibody against pAKT Ser-473 (4060) from Cell Signaling.

Techniques: Knockdown, Migration, Expressing, Transfection, Transwell Assay, Staining, Microscopy, shRNA, Control, Injection

Journal: The Journal of Biological Chemistry

Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

doi: 10.1074/jbc.M113.542688

Figure Lengend Snippet: Alteration of N-glycosylation in GOLPH3-knockdown cells. A, total expression levels of β1 integrin were analyzed by Western blotting (WB). The same amounts of cell lysates (200 μg) were obtained from control and KD cells, which were picked up from HeLa cells expressed as shRNA for control (Ctrl) and GOLPH3 (KD1,2) using the Phoenix system. Cell lysates were immunoprecipitated (IP) with anti-β1 antibody. The immunoprecipitates of β1 integrin were treated with (+) or without peptide:N-glycosidase F (PNGase) (−), and then immunoblotted with anti-β1 antibody (upper panel). The knockdown efficiency of GOLPH3 was confirmed by immunoblotting with anti-GOLPH3 antibody (middle panel). The α-tubulin was used as a loading control to warrant the same amounts of proteins to be used (lower panel). B, analysis of PA-N-glycans was by reversed-phase HPLC. Then N-glycans released from control or KD cells with peptide:N-glycosidase F were pyridylaminated as described under “Experimental Procedures.” The PA-N-glycans (upper panel), sequentially digested with sialidase (middle panel) and β-galactosidase (lower panel), were subjected to reversed phase HPLC. The asterisk indicates the peaks for sialylated N-glycans. C, RT-PCR for mRNA expression of several sialyltransferases and sialidase as indicated. The β-actin was used as a loading control. Ctrl, control shRNA; KD, shRNA for GOLPH3-knockdown; β1, integrin β1.

Article Snippet: The following antibodies were used: monoclonal antibodies against α5 (610634) and β1 (610468) from BD Biosciences; monoclonal antibody against α-tubulin (Sigma, T6199), β1 integrin for immunoprecipitation (TS2/16, ATCC); FACS or functional blocking (P5D2, DSHB); HA (Roche Applied Science, 1867423); antibody against FLAG for immunoblot (Sigma, F1804); immunoprecipitation (Sigma, A2220); polyclonal antibody against GOLPH3 (Abcam, ab82377); α3 integrin (Santa Cruz Biotechnology, Sc-6592); polyclonal antibody against EGFR (2232); AKT (9272); and monoclonal antibody against pAKT Ser-473 (4060) from Cell Signaling.

Techniques: Glycoproteomics, Knockdown, Expressing, Western Blot, Control, shRNA, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

doi: 10.1074/jbc.M113.542688

Figure Lengend Snippet: Effects of restoration of GOLPH3 expression on β1 integrin-mediated cell migration and its N-glycosylation. A, GOLPH3 knockdown cells as described in Fig. 2A were infected with retrovirus-expressing shRNA, a resistant GOLPH3 gene, and then cultured in the presence of hygromycin to achieve 100% infection, which was used as rescued cells (Res). Cell migration on laminin 332 (upper panel) was monitored by time-lapse microscopy as described under “Experimental Procedures.” Each bar represents the means ± S.D. of the migration distance of 10 cells in each assay (*, p < 0.0001, by one-way ANOVA with Tukey's post hoc test). The expression levels of GOLPH3 were compared among the control (Ctrl), the KD, and the rescued cells (middle panel), which were restored with the expression of GOLPH3 in KD cells. The α-tubulin was used as a loading control (lower panel). B, Western blotting for β1 integrin. The same amounts of cell lysates (200 μg) obtained from the indicated cells were immunoprecipitated (IP) with anti-β1 antibody, and the immunoprecipitates were digested with or without sialidase. After the treatment, the immunoprecipitates were then immunoblotted with β1 antibody. Ctrl, control shRNA; KD, GOLPH3-knockdown; Res, KD cells overexpressed a shRNA-resistant GOLPH3 gene. C, comparison of major N-glycans from different cells by glycan profiling using LC/MS peak area of asialo and sialylated N-glycans were calculated using mass spectra obtained in positive and negative ion modes, respectively. The relative peak area of major N-glycans from the control, KD, and the rescued cells were expressed as a percentage of the total peak area of the glycans. Glycan structures were deduced by MS analysis. D, proteins extracted from HeLa cells that expressed a Tet-On expression system for shRNA of GOLPH3-292 (left panel) or the GOLPH3 gene (right panel) using lentivirus systems as described under “Experimental Procedures” with or without doxycycline (DOX), were immunoprecipitated with the indicated lectin, resolved by SDS-PAGE, and then immunoblotted for β1 integrin antibody. Cell lysates were also Western blotted for the indicated antibodies. SSA is an α2,6-sialic acid-specific lectin, and MAM is an α2,3-specific lectin. E, HeLa cells that expressed Tet-inducible shRNA against GOLPH3-292 (KD; left panel) or GOLPH3 gene (OE; right panel) were propagated with (lower panel) or without (upper panel) 1 μg/ml doxycycline for 72 h, detached with trypsin/EDTA, and washed with DMEM, including 10% FBS. After washing the cells were stained with (solid line) or without (shadowed line) anti-β1 antibody (P5D2) and then stained with anti-mouse IgG conjugated with Alexa Fluor 647. The expression levels of β1 integrin on the cell surface were analyzed using BD FACSCalibur, operated with BD CellQuest Pro software. F, MDA-MB231 cells expressed with the Tet-On system for shRNA of GOLPH3-292 (KD1) or GOLPH3-442 (KD2) using the lentivirus were cultured in the presence (+) or absence (−) with 1 μg/ml doxycycline for 72 h. Cell lysates were immunoprecipitated with the SSA lectin, resolved by SDS-PAGE, and then immunoblotted for β1 integrin antibody. Cell lysates were also Western-blotted for the indicated antibodies. G, cell migration on FN was determined using a transwell assay in GOLPH3 knockdown cells as described in F. The quantitative data were obtained from three independent experiments. Data are presented as the means ± S.D. (**, p < 0.001 by one-way ANOVA with Tukey's post hoc test). H, HeLa cells that expressed Tet-inducible shRNA against GOLPH3-292 were propagated with (lower panel, KD cells) or without (upper panel, control cells) 1 μg/ml doxycycline for 72 h. The detached cells were incubated with MAM lectin, which recognizes α2,3-sialylation glycans (left panel), and with RCA-I lectin, which specifically recognizes terminal galactose residue (right panel) or without (a shadowed line), and then incubated with streptavidin conjugated with Alexa Fluor 647. The reactivities against MAM and RCA-I lectin were analyzed using BD FACSCalibur, operated with BD CellQuest Pro software. I– K indicated the changes of sialylation levels on integrin α5, α3, and EGFR, respectively, in GOLPH3 knockdown and overexpression cells as described in D. L, HeLa cells expressed with the Tet-On system for overexpression of GOLPH3 as described in D were cultured in the presence (+) or absence (−) of doxycycline (DOX) at 1 μg/ml for 72 h. The cell migration on FN was determined using a transwell assay. Data are presented as the means ± S.D.; **, p < 0.01 by one-tail unpaired t test.

Article Snippet: The following antibodies were used: monoclonal antibodies against α5 (610634) and β1 (610468) from BD Biosciences; monoclonal antibody against α-tubulin (Sigma, T6199), β1 integrin for immunoprecipitation (TS2/16, ATCC); FACS or functional blocking (P5D2, DSHB); HA (Roche Applied Science, 1867423); antibody against FLAG for immunoblot (Sigma, F1804); immunoprecipitation (Sigma, A2220); polyclonal antibody against GOLPH3 (Abcam, ab82377); α3 integrin (Santa Cruz Biotechnology, Sc-6592); polyclonal antibody against EGFR (2232); AKT (9272); and monoclonal antibody against pAKT Ser-473 (4060) from Cell Signaling.

Techniques: Expressing, Migration, Glycoproteomics, Knockdown, Infection, shRNA, Cell Culture, Time-lapse Microscopy, Control, Western Blot, Immunoprecipitation, Comparison, Liquid Chromatography with Mass Spectroscopy, SDS Page, Staining, Software, Transwell Assay, Incubation, Residue, Over Expression

Journal: The Journal of Biological Chemistry

Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

doi: 10.1074/jbc.M113.542688

Figure Lengend Snippet: Forced expression of ST6GAL1 led to a restoration of the cell migration and the phosphorylation of AKT, which were suppressed in the GOLPH3-knockdown cells. The cell migration was examined by using the Boyden chamber described under “Experimental Procedures” in the presence (+) or absence (−) of doxycycline (DOX) at 1 μg/ml. The siRNAs of GOLPH3 and control (Ctrl) were transiently transfected into cells containing the Tet-On expression system for ST6GAL1 (A) or ST3GAL4 (B). Migrated cells were then counted under a microscope. A representative example is shown in the left panel. The scale bar, 100 μm. The quantitative data were obtained from three independent experiments (right panel). Results are expressed as the mean number of cells migrated ± S.D. (**, p < 0.001 by one-way ANOVA with Tukey's post hoc test, n = 5). C, cells that expressed inducible shRNA against GOLPH3 (left panel), with or without the forced expression of ST6GAL1 (right panel), were cultured in the presence (+) or absence (−) of DOX at 1 μg/ml for 72 h and then treated with (+) or without (−) EGF at 5 ng/ml for 2 or 5 min. Immunoblot analysis was performed with the indicated antibodies. D, HeLa cells that were expressed with Tet-On expression systems for both shRNAs of GOLPH3-292 and the ST6GAL1-FLAG genes were cultured in the presence (right lane) or absence (left lane) of DOX at 1 μg/ml for 72 h. The cell lysates were immunoprecipitated (IP) with SSA lectin. The immunoprecipitates and cell lysates were resolved on SDS-PAGE and blotted with several antibodies as indicated. E, inhibition of the mTOR signaling pathway by rapamycin induced the expression of sialylated β1 integrin. Proteins extracted from MDA-MB231 cells, which were treated with the indicated concentration of rapamycin for 72 h, were immunoprecipitated with SSA-agarose or MAM-agarose, resolved by SDS-PAGE, and then immunoblotted for β1 integrin antibody. Cell lysates were also Western blotted for the indicated antibody.

Article Snippet: The following antibodies were used: monoclonal antibodies against α5 (610634) and β1 (610468) from BD Biosciences; monoclonal antibody against α-tubulin (Sigma, T6199), β1 integrin for immunoprecipitation (TS2/16, ATCC); FACS or functional blocking (P5D2, DSHB); HA (Roche Applied Science, 1867423); antibody against FLAG for immunoblot (Sigma, F1804); immunoprecipitation (Sigma, A2220); polyclonal antibody against GOLPH3 (Abcam, ab82377); α3 integrin (Santa Cruz Biotechnology, Sc-6592); polyclonal antibody against EGFR (2232); AKT (9272); and monoclonal antibody against pAKT Ser-473 (4060) from Cell Signaling.

Techniques: Expressing, Migration, Phospho-proteomics, Knockdown, Control, Transfection, Microscopy, shRNA, Cell Culture, Western Blot, Immunoprecipitation, SDS Page, Inhibition, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: An Oncogenic Protein Golgi Phosphoprotein 3 Up-regulates Cell Migration via Sialylation *

doi: 10.1074/jbc.M113.542688

Figure Lengend Snippet: Proposed molecular mechanism for the regulation of sialylation and cellular signaling by GOLPH3. GOLPH3 has been known to modulate mTOR signaling (21). This study clearly showed that the interaction among GOLPH3, PI4P, and sialyltransferases might efficiently regulate α2,6-sialylation on several target proteins expressed on the cell surface, including integrins and some receptor tyrosine kinases (RTK). Then those resultant glycoproteins could cooperatively enhance integrin-mediated cell migration and activate cellular signal pathways such as the phosphoinositide 3-kinase (PI3K)-AKT-mTOR cascade. The possible molecular mechanism described here might partly explain the observation that the GOLPH3 gene is usually amplified in many malignant tumors.

Article Snippet: The following antibodies were used: monoclonal antibodies against α5 (610634) and β1 (610468) from BD Biosciences; monoclonal antibody against α-tubulin (Sigma, T6199), β1 integrin for immunoprecipitation (TS2/16, ATCC); FACS or functional blocking (P5D2, DSHB); HA (Roche Applied Science, 1867423); antibody against FLAG for immunoblot (Sigma, F1804); immunoprecipitation (Sigma, A2220); polyclonal antibody against GOLPH3 (Abcam, ab82377); α3 integrin (Santa Cruz Biotechnology, Sc-6592); polyclonal antibody against EGFR (2232); AKT (9272); and monoclonal antibody against pAKT Ser-473 (4060) from Cell Signaling.

Techniques: Migration, Amplification

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Representative images of cell clusters in the vasculature of human primary breast tumor. The distributions of CD206 (brown), CD44 (brown), CK (brown), and CD31 (red, indicating vessels) in tissue serial sections from luminal-like BrCa ( n = 15) were determined by immunohistochemical staining. Nuclei are stained with hematoxylin. B Immunohistochemical staining of CD206, CD44, and CD31 was performed in serial tissue sections of human BrCa. Tumors with and without microemboli from a tissue microarray (85 human breast lesions) were analyzed. Using Image-Pro Plus software, the expression intensity of each molecule was evaluated by integral optical density and compared with and without microemboli. Data were analyzed with the Mann-Whitney test. * p < 0.05, *** p < 0.001. C The expression of CD44 and CD206 in the invasion front versus non-invasive front of serial sections from human BrCa tissues was analyzed. Data represents the mean ± SEM. Mann-Whitney test. D A positive association between high frequency of CD206 + -macrophage and CD44 high -cancer cells in BrCa.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Immunohistochemical staining, Staining, Microarray, Software, Expressing, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Adaptive CD44 expression of luminal-like BrCa cells (MCF7 and T47D) upon THP-1-derived M2-like macrophages stimulation in a non-contact transwell system was assessed by immunoblotting analysis. GAPDH was used as control. The band intensities were analyzed by densitometry analysis. * p < 0.05. B Representative confocal images of CD44 expression in disseminated cell clusters on top-3D matrix membrane during collective detachment. DAPI was used to stain the nuclei. An acquired CD44 high state upon TAMs stimulation in primary BrCa cell clusters (purified from MMTV-PyMT tumor) was observed by immunofluorescence analysis. C Co-immunoprecipitation experiment from whole-cell extracts demonstrating the interaction between CD44 and Ezrin after co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system. The band intensities were analyzed by densitometry analysis. * p < 0.05. D The influence of CD44 knockdown on collective invasion of cell clusters in 3D matrix membrane. The sh-Control and sh-CD44 primary BrCa cell clusters, premixed with or without TAMs, were embedded in 3D system and recorded by time-lapse microscopy. TAMs were pre-stained with Vybrant CM-DiI (red). E Representative confocal images of localization of pEzrin, pMyosin, β-catenin and ZO-1 at the invasive and disseminating clusters. The sh-Control and sh-CD44 of primary BrCa cell clusters, premixed with or without TAMs, were cultured in top-3D basement. F Knockdown of CD44 inhibited TAMs-induced formation of mammosphere. The mammosphere-forming capacity of primary BrCa cells purified from MMTV-PyMT tumors was detected. Primary BrCa cells were directly sorted into wells of ultra-low attachment 24-well plate containing mammosphere growth medium, and then co-cultured with or without TAMs for 12 days in a non-contact transwell system. G Representative intravital images of subcutaneous xenografts derived from MCF7/sh-Control/GFP + and MCF7/sh-CD44/GFP + cells in vivo, which were injected with or without THP-1-derived M2-like macrophages, showing cohesive-invading MCF7/GFP + cells approaching around vascular vessel. Tomato lectin (DyLight649, Cat. L32472, Thermo Fisher) was injected via tail vein to label vascular structures. Scale bars, 100 μm. See Supplementary Video – . H Representative multiphoton confocal images of thick tumor tissues showed collective dissemination in subcutaneous xenografts. Tumor tissues were cut into 10- to 12-µm thick for paraffin sections, and the blood vessels were labeled by CD31 (Red channel) using immunohistochemical staining. Scale bars, 100 μm. See Supplementary Video – .

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing, Derivative Assay, Western Blot, Control, Membrane, Staining, Purification, Immunofluorescence, Immunoprecipitation, Cell Culture, Knockdown, Time-lapse Microscopy, In Vivo, Injection, Labeling, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A The mRNA levels of a panel of cytokines in TAMs versus PBMCs isolated from five primary breast tumors (MMTV-PyMT) were detected by qRT-PCR. B The intracellular CCL8 and IL-10 in TAMs versus PBMCs isolated from normal mouse or tumor-bearing mouse (MMTV-PyMT tumor) were determined by ELISA. n = 5/group. Bars correspond to mean ± SD. * p < 0.05, ** p < 0.01, ns, nonsignificant. C The secreted CCL8 and IL-10 in TAMs versus primary BrCa cells were determined by ELISA. Cells were isolated from five primary breast tumors (MMTV-PyMT) and cultured separately. Bars correspond to mean ± SD. ** p < 0.01, ns, nonsignificant. D Fluorescence in situ hybridization analysis of human BrCa tissue sections revealed that CCL8 mRNA is found in CD206 + TAMs but not in cancer cells. The expression of CD206 was detected using immunochemical staining. Inset representing a CD206 + macrophage-expressing CCL8 mRNA. Scale bars, 100 μm ( n = 3). E Blocking of CCL8 inhibited the collective migration induced by THP-1-derived M2-like macrophages. In vitro scratch assay of untreated MCF7 or treated with THP-1-derived M2-like macrophages, THP-1-derived M2-like macrophages plus normal IgG, or THP-1-derived M2-like macrophages plus CCL8 antibody for the indicated period of time. Red line, cell culture margins ( n = 4). F Blocking of CCL8 inhibited the re-distribution of invadopodia molecules in collective migration induced by THP-1-derived M2-like macrophages. Representative confocal images showed the re-distribution of Cortactin (red) and TKS5 (green) at the migrating front of clusters after CCL8 blocking for 3 days. MCF7 cells co-cultured with THP-1-derived M2-like macrophages in a non-contact transwell system, followed by treatment with normal IgG or CCL8 antibody. Scale bars, 20 μm. G Knocking down of CCL8 in THP-1-derived M2-like macrophages inhibited the increase of Vinculin at the migrating front of clusters. Wound healing assay was used to observe the changes of Vinculin expression at the migrating front. M2-like macrophages derived from THP-1 were transfected with siRNAs (si-Control or si-CCL8) and then co-cultured with MCF7 cells in a non-contact transwell system for 3 days. The expression of Vinculin was determined by immunofluorescence assay. Scale bars, 10 μm. H Blocking of CCL8 inhibited the onset of xenografts induced by THP-1-derived M2-like macrophages in vivo. MCF7 cells were orthotopically implanted into mammary pad of nu/nu mice, followed by intraperitoneal administration of a neutralizing antibody for CCL8. I HE staining of MCF7 xenografts in normal IgG or CCL8 antibody-treated mice. J CD44 (brown) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice. K CD31 (red) expression of MCF7 xenografts in normal IgG- or CCL8 antibody-treated mice.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Fluorescence, In Situ Hybridization, Expressing, Staining, Blocking Assay, Migration, Derivative Assay, In Vitro, Wound Healing Assay, Transfection, Control, Immunofluorescence, In Vivo

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A The influence of CCL8 on the expression of CD44 in MCF7 cells was evaluated by western blot. The band intensities were analyzed by densitometry analysis. * p < 0.05. B Blocking of CCL8 inhibited the acquisition of CD44 high state in MCF7 cells induced by M2-like macrophages, which was obtained from human monocytic cell line THP-1 stimulated by PMA/m-CSF/IL-4/IL-10/IL-13. The band intensities were analyzed by densitometry analysis. ** p < 0.01, ns: no significance. C Scheme of identifying differential CD44-interacting peptides upon THP-1-derived M2-like macrophages/CCL8 stimulation. The intersection showed the CD44-interacting proteins, which were analyzed by co-immunoprecipitation-based mass spectrometry assay. MCF7 and T47D cells were stimulated by THP-1-derived M2-like macrophages or CCL8 for 3 days, respectively. The proteins recruited to CD44 were precipitated by CD44 antibody, and then subject to mass spectrometry assay. D Functional associations of the regulatory networks of MDM2/p53-correlated genes from analysis of STRING data are presented. E The effects of CCL8 on the activation of MDM2 in MCF7 cells were evaluated by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05. F Blocking of CCL8 inhibited the activation of MDM2 in MCF7 cells induced by THP1-derived M2-like macrophages. The band intensities were analyzed by densitometry analysis. * p < 0.05, ** p < 0.01, ns: no significance. G Repression of p53 expression by M2-like macrophages in human BrCa cells was detected by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05. H Knocking down of MDM2 in primary BrCa cells inhibited the up-regulation of CD44 induced by TAMs. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. I The influence of MDM2 inhibitor on collective dissemination of cell clusters induced by TAMs in 2D culture system. Before co-cultured with TAMs on 2D culture system, primary BrCa cell clusters were obtained from an ultra-low attachment culture system. TAMs were pre-stained with Vybrant CM-DiI (red).

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Blocking Assay, Derivative Assay, Immunoprecipitation, Mass Spectrometry, Functional Assay, Activation Assay, Cell Culture, Staining

Journal: Cell Death & Disease

Article Title: Activation of CD44 signaling in leader cells induced by tumor-associated macrophages drives collective detachment in luminal breast carcinomas

doi: 10.1038/s41419-022-04986-4

Figure Lengend Snippet: A Activation of Ezrin and p38 after MCF7 cells co-cultured with THP-1-derived M2-like macrophages or CCL8 by a non-contact transwell system was determined by immunoblotting assay. The band intensities were analyzed by densitometry analysis. * p < 0.05, *** p < 0.001. B Knocking down of CD44 inhibited the activation of Ezrin and p38 induced by THP-1-derived M2-like macrophages or CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. C Knocking down of Ezrin in primary BrCa cells inhibited collective dissemination induced by TAMs in 2D culture system. The primary BrCa cell clusters were transfected by si-Ezrin. TAMs were pre-stained with Vybrant CM-DiI (red). D The p38 inhibitor attenuated collective dissemination of primary BrCa cell clusters induced by TAMs. TAMs were prestained with Vybrant CM-DiI (red). E Knocking down of Ezrin inhibited phosphorylation of Cortactin and p38 triggered by CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. F Knocking down of p38 repressed the phosphorylation of Cortactin induced by CCL8. The band intensities were analyzed by densitometry analysis. *** p < 0.001, ns: no significance. G Scheme summarizing the proposed mechanism by which MDM2-p53-p38 signaling pathway mediated the acquisition of CD44 high state, which leading to cohesive detachment.

Article Snippet: The mouse CD44 shRNA lentiviral particles (sc-35534-V) were purchased from Santa Cruz (Dallas, TX, USA).

Techniques: Activation Assay, Cell Culture, Derivative Assay, Western Blot, Transfection, Staining, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

doi: 10.1074/jbc.m111.327296

Figure Lengend Snippet: FIGURE 1. Cancer-associated BRCT mutations prevent BRCA1 centrosome localization. Shown is a schematic diagram showing the organization of BRCA1 protein domains (the RING and BRCT domains, the NLS, and the NES) as well as cancer-associated mutations. YFP-tagged BRCA1 cancer-associated mutants were transfected into MCF-7 breast cancer cells and analyzed for co-localization with the centrosome-component -tubulin by immunofluorescence micros- copy. Representative cell images of YFP-BRCA1 localization are shown in the right-hand panel, in addition to close-up images of the centrosomes, with staining of BRCA1 and -tubulin. Cells expressing YFP-tagged BRCA1 were scored for co-localization with the centrosome. Scoring results were obtained from at least three independent experiments, each with at least 100 cells scored (mean S.D. (error bars)). Integrity of the BRCA1 point mutants was validated by Western blot (see inset).

Article Snippet: Immunofluorescence Microscopy—Centrosomal protein immunostaining was performed as described previously (38), fixing cells with acetone/methanol and staining cells with the following primary antibodies: rabbit polyclonal BRCA1 Ab-P (1:1000; gift from Prof. Jeffrey Parvin), mouse monoclonal -tubulin (1:1000; Abcam), mouse monoclonal -tubulin (1:1000; Sigma), and rabbit polyclonal anti-FLAG (1:2000; Sigma).

Techniques: Transfection, Immunofluorescence, Staining, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

doi: 10.1074/jbc.m111.327296

Figure Lengend Snippet: FIGURE 2. Mapping of BRCA1 centrosome targeting domains. A, diagram showingthepositionofBRCA1proteindomains:theRINGandBRCTdomains, NLS, NES, and the binding regions for BARD1 and -tubulin. A range of YFP- tagged BRCA1 deletions and fusions were transiently expressed in MCF-7 cells and then fixed with acetone/methanol and analyzed for co-localization of BRCA1 with -tubulin by immunofluorescence microscopy (as detailed in the legend to Fig. 1). The relative degree of centrosome-positive BRCA1 stain- ing is indicated in the right-hand panel. B, pYFP-BRCA1-transfected cells were scored for centrosomal localization of ectopic BRCA1 sequences by fluores- cence microscopy, and the data were graphed as shown. Scoring data are from three independent experiments, scoring at least 100 cells/experiment

Article Snippet: Immunofluorescence Microscopy—Centrosomal protein immunostaining was performed as described previously (38), fixing cells with acetone/methanol and staining cells with the following primary antibodies: rabbit polyclonal BRCA1 Ab-P (1:1000; gift from Prof. Jeffrey Parvin), mouse monoclonal -tubulin (1:1000; Abcam), mouse monoclonal -tubulin (1:1000; Sigma), and rabbit polyclonal anti-FLAG (1:2000; Sigma).

Techniques: Binding Assay, Immunofluorescence, Microscopy, Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

doi: 10.1074/jbc.m111.327296

Figure Lengend Snippet: FIGURE 3. Centrosome targeting domains of BRCA1 are insufficient for the regulation of centrosome amplification. A, diagram of different BRCA1 peptides tested for their ability to regulate centrosome amplification (including the minimal centrosomal targeting sequence) and their main functional defects. B, YFP-tagged BRCA1 proteins were transiently expressed in HCC1937 breast cancer cells, which harbor the endogenous BRCA1 mutation 5382InsC and have a defective DNA damage checkpoint. Cells were treated with 10 Gy of IR and left to recover for 48 h. After fixation with acetone/methanol, cells were immunostained with anti--tubulin antibody and scored by microscopy for the number of cells displaying centrosome amplification (2 centrosomes/cell). Representative cell images are shown for each BRCA1 peptide, showing similar expression levels of transiently expressed proteins and close up images of centrosomes. The number of centrosomes in each imaged cell is noted in white. C, IR-induced centrosome amplification was reduced by overexpression of WT BRCA1 but not by various mutated forms of BRCA1, including the minimal targeting sequence (N304C243). Scoring results were obtained from three independent experiments with at least 100 cells scored (mean S.D. (error bars)). Student’s t test was used to determine that only YFP-BRCA1 wild type was statistically significant in regulating centrosome amplification in comparison with YFP control. ***, p 0.001.

Article Snippet: Immunofluorescence Microscopy—Centrosomal protein immunostaining was performed as described previously (38), fixing cells with acetone/methanol and staining cells with the following primary antibodies: rabbit polyclonal BRCA1 Ab-P (1:1000; gift from Prof. Jeffrey Parvin), mouse monoclonal -tubulin (1:1000; Abcam), mouse monoclonal -tubulin (1:1000; Sigma), and rabbit polyclonal anti-FLAG (1:2000; Sigma).

Techniques: Amplification, Sequencing, Functional Assay, Mutagenesis, Microscopy, Expressing, Over Expression, Comparison, Control

Journal: Journal of Biological Chemistry

Article Title: Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

doi: 10.1074/jbc.m111.327296

Figure Lengend Snippet: FIGURE 4. Defining the dynamics of YFP-BRCA1 turnover and retention at the centrosome in live cells. A, FRAP analysis was performed on MCF-7 cells transfected with plasmids encoding RFP-pericentrin C241 and YFP-BRCA1 (wild type or N304C243 mutant). Centrosomes were targeted for laser photo- bleaching followed by fluorescence time lapse microscopy. Representative prebleach, first image postbleach, and images for 10.5, 18.5, and 26.5 s after bleach are shown for each construct as well as the marker for the centrosome, RFP-pericentrin C241. The insets show higher magnification views of the centrosome. Corresponding FRAP recovery curves are shown for each protein in comparison with RFP-pericentrin C241 (recently described in Ref. 37), indicating the immobile and mobile fractions (left). YFP-BRCA1 (wild type) and RFP-pericentrin were also analyzed by an inverse FRAP (iFRAP) assay, bleaching cellular fluorescence,andthenquantifyingtherateoflossfromthecentrosome(right).B,thet1⁄2(half-timeS.E.(errorbars))andimmobilefraction(percentageS.E.) are shown for each protein with an average of 10–15 cells over at least two experiments analyzed for each. Student’s t test was used to show a significant difference in t1⁄2 between wild-type and N304C243 peptides. **, p 0.01. C, in an in vitro assay to measure BRCA1 centrosomal retention, cells were treated with CSK detergent buffer for 0, 8, or 40 min prior to fixation with acetone/methanol to remove soluble protein. Cells were then immunostained with BRCA1 antibody, co-stained for -tubulin, and scored by microscopy for the percentage of cells still displaying endogenous BRCA1 at the centrosome.

Article Snippet: Immunofluorescence Microscopy—Centrosomal protein immunostaining was performed as described previously (38), fixing cells with acetone/methanol and staining cells with the following primary antibodies: rabbit polyclonal BRCA1 Ab-P (1:1000; gift from Prof. Jeffrey Parvin), mouse monoclonal -tubulin (1:1000; Abcam), mouse monoclonal -tubulin (1:1000; Sigma), and rabbit polyclonal anti-FLAG (1:2000; Sigma).

Techniques: Transfection, Mutagenesis, Fluorescence, Time-lapse Microscopy, Construct, Marker, Comparison, In Vitro, Staining, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

doi: 10.1074/jbc.m111.327296

Figure Lengend Snippet: FIGURE 5. CRM1 contributes to BRCA1 centrosomal localization. A, YFP- CRM1 and RFP-pericentrin C241 were transiently co-expressed to show that ectopic CRM1 localizes to the centrosome in live MCF-7 cells. B, MCF-7 cells were treated with 5 ng/ml LMB for 12 h to inhibit CRM1 binding to BRCA1. After fixation with acetone/methanol, cells were stained with anti--tubulin and anti-BRCA1 Ab-P antibodies, and the relative levels of fluorescence inten- sity at the centrosomes were analyzed by microscopy. 200 cells were ana- lyzed (mean S.D. (error bars)). Student’s t test was used to show that the loss of BRCA1 staining at the centrosome after LMB was statistically significant. ***, p 0.001. C, MCF-7 cells were transfected with plasmids encoding wild- type BRCA1 with or without LMB, and nuclear transport mutants of BRCA1 were then scored for co-localization with -tubulin. Representative images of each construct are shown with centrosomal localization indicated. Scoring results are from three experiments (mean S.D.). Student’s t test was used to determine the statistical significance of ectopic BRCA1 loss from the centro- some when CRM1-binding was inhibited by LMB or NES mutation. **, p 0.01; ***, p 0.001.

Article Snippet: Immunofluorescence Microscopy—Centrosomal protein immunostaining was performed as described previously (38), fixing cells with acetone/methanol and staining cells with the following primary antibodies: rabbit polyclonal BRCA1 Ab-P (1:1000; gift from Prof. Jeffrey Parvin), mouse monoclonal -tubulin (1:1000; Abcam), mouse monoclonal -tubulin (1:1000; Sigma), and rabbit polyclonal anti-FLAG (1:2000; Sigma).

Techniques: Binding Assay, Staining, Fluorescence, Microscopy, Transfection, Construct, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Characterization of BRCA1 Protein Targeting, Dynamics, and Function at the Centrosome

doi: 10.1074/jbc.m111.327296

Figure Lengend Snippet: FIGURE 6. NES mutation disrupts BRCA1 dynamics and function at the centrosome. A, FRAP analysis was performed on live MCF-7 cells co-transfected with RFP-pericentrin C241 and YFP-BRCA1 mutants, 70–1863 and NESm, deficient in binding to BARD1 and CRM1, respectively. The 70–1863 sequence is highly export-active (39). Centrosome fluorescence was photobleached, and fluorescence recovery was then measured by time lapse microscopy. Representative prebleach, first image postbleach, and images for 10.5, 18.5, and 26.5 s after bleach are shown for each protein. The insets show higher magnification views of thetargetarea.Correspondingrecoverycurvesareshownfortheaboveproteins,aswellaswild-typeBRCA1.Thet1⁄2(half-timeS.E.(errorbars))andimmobile fractions(percentageS.E.)arealsoshown.Statisticalsignificanceofchangesinimmobilefractionandhalf-timeareindicated.*,p0.05;***,pvalue0.001. B,differentBRCA1mutantsdefectiveinbindingtoBARD1orinnucleartransportweretestedfortheirabilitytoregulatecentrosomalamplification.YFP-tagged BRCA1 proteins were transfected into HCC1937 breast cancer cells, and cells were then treated with 10 Gy of IR and left to recover for 72 h. Cells were fixed and immunostained with anti--tubulin antibody and analyzed for centrosome amplification (2 centrosomes/cell). Scoring results are from three independent experiments (mean S.D.) and Student’s t test was used to determine statistical significance. **, p value 0.01; ***, p value 0.001.

Article Snippet: Immunofluorescence Microscopy—Centrosomal protein immunostaining was performed as described previously (38), fixing cells with acetone/methanol and staining cells with the following primary antibodies: rabbit polyclonal BRCA1 Ab-P (1:1000; gift from Prof. Jeffrey Parvin), mouse monoclonal -tubulin (1:1000; Abcam), mouse monoclonal -tubulin (1:1000; Sigma), and rabbit polyclonal anti-FLAG (1:2000; Sigma).

Techniques: Mutagenesis, Transfection, Binding Assay, Sequencing, Fluorescence, Time-lapse Microscopy, Amplification