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ductal carcinoma cell line mda mb 435s (ATCC)

Name: ductal carcinoma cell line mda mb 435s
Brand: ATCC
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ATCC ductal carcinoma cell line mda mb 435s

Analysis of EGFR expression levels and binding affinities of sdAbs to human EGFR-presenting cells. Whole-cell lysates of exponentially growing cells (A431, FaDu, <t>MDA-MB</t> <t>435S)</t> were prepared and equal amounts of total cellular proteins were separated by SDS-PAGE on 10% polyacrylamide gels. After Western Blot transfer onto PVDF membranes, EGFR and β-actin proteins were detected by incubation with the respective specific antibodies followed by HRP-coupled antibodies and chemiluminescence detection (A) . In vitro specificity of 99m Tc-7C12 and 99m Tc-EG2 on A431 and FaDu cells was investigated after 1 h incubation on ice (B) . Binding of radiolabeled sdAbs was blocked by 40-fold excess of unlabeled Cetuximab. Binding data is expressed as percent of injected dose per mg protein (% ID/mg protein). NB = non-blocked, B = blocked. For in vitro binding studies, two dimensional cultures of A431, FaDu and MDA-MB 435S cells were incubated with increasing concentrations of 99m Tc-7C12 (C) . Total binding was measured in the absence of and nonspecific binding in the presence of 1 mM unlabeled sdAb. Specific binding was calculated as the difference between total and nonspecific binding. Binding studies were repeated twice and representative saturation curves for the EGFR-positive cell lines A431 and FaDu are shown. For the EGFR-negative cell line MDA-MB 435S, no specific binding was observed.

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1) Product Images from "High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli"

Article Title: High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-12-97

Figure Legend Snippet: Analysis of EGFR expression levels and binding affinities of sdAbs to human EGFR-presenting cells. Whole-cell lysates of exponentially growing cells (A431, FaDu, MDA-MB 435S) were prepared and equal amounts of total cellular proteins were separated by SDS-PAGE on 10% polyacrylamide gels. After Western Blot transfer onto PVDF membranes, EGFR and β-actin proteins were detected by incubation with the respective specific antibodies followed by HRP-coupled antibodies and chemiluminescence detection (A) . In vitro specificity of 99m Tc-7C12 and 99m Tc-EG2 on A431 and FaDu cells was investigated after 1 h incubation on ice (B) . Binding of radiolabeled sdAbs was blocked by 40-fold excess of unlabeled Cetuximab. Binding data is expressed as percent of injected dose per mg protein (% ID/mg protein). NB = non-blocked, B = blocked. For in vitro binding studies, two dimensional cultures of A431, FaDu and MDA-MB 435S cells were incubated with increasing concentrations of 99m Tc-7C12 (C) . Total binding was measured in the absence of and nonspecific binding in the presence of 1 mM unlabeled sdAb. Specific binding was calculated as the difference between total and nonspecific binding. Binding studies were repeated twice and representative saturation curves for the EGFR-positive cell lines A431 and FaDu are shown. For the EGFR-negative cell line MDA-MB 435S, no specific binding was observed.

Techniques Used: Expressing, Binding Assay, SDS Page, Western Blot, Incubation, In Vitro, Injection

Image Search Results

Journal: Microbial Cell Factories

Article Title: High-yield production of functional soluble single-domain antibodies in the cytoplasm of Escherichia coli

doi: 10.1186/1475-2859-12-97

Figure Lengend Snippet: Analysis of EGFR expression levels and binding affinities of sdAbs to human EGFR-presenting cells. Whole-cell lysates of exponentially growing cells (A431, FaDu, MDA-MB 435S) were prepared and equal amounts of total cellular proteins were separated by SDS-PAGE on 10% polyacrylamide gels. After Western Blot transfer onto PVDF membranes, EGFR and β-actin proteins were detected by incubation with the respective specific antibodies followed by HRP-coupled antibodies and chemiluminescence detection (A) . In vitro specificity of 99m Tc-7C12 and 99m Tc-EG2 on A431 and FaDu cells was investigated after 1 h incubation on ice (B) . Binding of radiolabeled sdAbs was blocked by 40-fold excess of unlabeled Cetuximab. Binding data is expressed as percent of injected dose per mg protein (% ID/mg protein). NB = non-blocked, B = blocked. For in vitro binding studies, two dimensional cultures of A431, FaDu and MDA-MB 435S cells were incubated with increasing concentrations of 99m Tc-7C12 (C) . Total binding was measured in the absence of and nonspecific binding in the presence of 1 mM unlabeled sdAb. Specific binding was calculated as the difference between total and nonspecific binding. Binding studies were repeated twice and representative saturation curves for the EGFR-positive cell lines A431 and FaDu are shown. For the EGFR-negative cell line MDA-MB 435S, no specific binding was observed.

Article Snippet: For binding and uptake studies, three different adherent human tumor cell lines were used: the epidermoid carcinoma cell line A431 (ATCC® Number: CRL-1555), the squamous cell carcinoma cell line FaDu (ATCC® Number: HTB-43), the ductal carcinoma cell line MDA-MB 435S (ATCC® Number: HTB-129).

Techniques: Expressing, Binding Assay, SDS Page, Western Blot, Incubation, In Vitro, Injection

A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from MDA-MB-435S cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.

Journal: PLoS ONE

Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

doi: 10.1371/journal.pone.0080171

Figure Lengend Snippet: A . Experion Bioanalyser gel image for RNA isolated following immunoprecipitation with YB-1 antibody or IgG isotype control antibody (labelled YB-1 IP and IgG IP respectively) from MDA-MB-435S cells. Ribosomal RNAs (18S and 28S) are shown by arrows. The ladder shows the size of the RNAs in nucleotides. Small RNAs are highlighted by the labelled bracket. B . RT-qPCR detection of mRNAs bound following immunoprecipitation with the YB-1 antibody.

Article Snippet: MCF7 and MDA-MB-435S breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Isolation, Immunoprecipitation, Quantitative RT-PCR

sncRNAs that are bound by YB-1 protein by immunoprecipitation in MCF7 and MDA-MB-435S cells based on enrichment in abundance in IP over input.

Journal: PLoS ONE

Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

doi: 10.1371/journal.pone.0080171

Figure Lengend Snippet: sncRNAs that are bound by YB-1 protein by immunoprecipitation in MCF7 and MDA-MB-435S cells based on enrichment in abundance in IP over input.

Article Snippet: MCF7 and MDA-MB-435S breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

Techniques: Immunoprecipitation

A. MCF7 cells B. MDA-MB-435S cells Note: miR-886 abundance in MCF7 cells was undetectable.

Journal: PLoS ONE

Article Title: Links between the Oncoprotein YB-1 and Small Non-Coding RNAs in Breast Cancer

doi: 10.1371/journal.pone.0080171

Figure Lengend Snippet: A. MCF7 cells B. MDA-MB-435S cells Note: miR-886 abundance in MCF7 cells was undetectable.

Article Snippet: MCF7 and MDA-MB-435S breast cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA).

Techniques:

Quantitative analysis of the ratio of phosphorylated to total OPN in the CM of cancer cells. The CM of A549, H460, and MDA-MB435S cancer cells were treated with or without calf intestine alkaline phosphatase (CIAP) and were then subjected to phos-tag ELISA. Two-tailed unpaired Student’s t -test, mean ± SEM of three independent assays conducted in triplicate.

Journal: Biomolecules

Article Title: Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility

doi: 10.3390/biom11091323

Figure Lengend Snippet: Quantitative analysis of the ratio of phosphorylated to total OPN in the CM of cancer cells. The CM of A549, H460, and MDA-MB435S cancer cells were treated with or without calf intestine alkaline phosphatase (CIAP) and were then subjected to phos-tag ELISA. Two-tailed unpaired Student’s t -test, mean ± SEM of three independent assays conducted in triplicate.

Article Snippet: H358 (human lung cancer), H460 (human lung cancer), and MDA-MB435S (human melanoma) cell lines were obtained from ATCC.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test

Relationship between OPN phosphorylation and OPN-induced cancer cell migration. ( a ) Effect of H460 CM and MDA-MB435S CM on cell migration of H358 cells. PBS was used as a sample instead of CM in the control experiments. One-way ANOVA and Tukey post-hoc test, mean ± SEM of three independent assays conducted in triplicate. ( b ) Effect of treatment of A549 and H460 CM with phosphatase (CIAP) on the A549 and H460 CM-induced cell migration in H358 and MIAPaca-2 cells. – and + indicate CM without and with CIAP treatment, respectively. Two-tailed unpaired Student’s t -test, mean ± SEM of three independent assays conducted in triplicate.

Journal: Biomolecules

Article Title: Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility

doi: 10.3390/biom11091323

Figure Lengend Snippet: Relationship between OPN phosphorylation and OPN-induced cancer cell migration. ( a ) Effect of H460 CM and MDA-MB435S CM on cell migration of H358 cells. PBS was used as a sample instead of CM in the control experiments. One-way ANOVA and Tukey post-hoc test, mean ± SEM of three independent assays conducted in triplicate. ( b ) Effect of treatment of A549 and H460 CM with phosphatase (CIAP) on the A549 and H460 CM-induced cell migration in H358 and MIAPaca-2 cells. – and + indicate CM without and with CIAP treatment, respectively. Two-tailed unpaired Student’s t -test, mean ± SEM of three independent assays conducted in triplicate.

Article Snippet: H358 (human lung cancer), H460 (human lung cancer), and MDA-MB435S (human melanoma) cell lines were obtained from ATCC.

Techniques: Migration, Two Tailed Test

Induction of cancer cell migration by phosphorylated OPN. ( a ) Western blot analysis of phosphorylation using phos-tag in a recombinant OPN after kinase treatment. The unprocessed blot image is shown in . ( b ) Cell migration of H358 cells in the presence or absence of recombinant OPN pretreated with kinase and OPN function-blocking antibody (OPN Ab) either separately or in combinations. One-way ANOVA and Tukey post-hoc test, mean ± SEM of three independent assays conducted in triplicate. ( c ) Phos-tag ELISA analysis of phosphorylated OPN in MDA-MB435S CM after kinase treatment. Two-tailed unpaired Student’s t -test, mean ± SEM of three independent assays conducted in triplicate. ( d ) Cell migration of H358 cells in the presence or absence of the MDA-MB435S CM pretreated with kinase and OPN function-blocking antibody (OPN Ab), either separately or in combination. – and + indicate treatment without and with the indicated reagent. One-way ANOVA and Tukey post-hoc test, mean ± SEM of three independent assays conducted in triplicate.

Journal: Biomolecules

Article Title: Phosphorylated Osteopontin Secreted from Cancer Cells Induces Cancer Cell Motility

doi: 10.3390/biom11091323

Figure Lengend Snippet: Induction of cancer cell migration by phosphorylated OPN. ( a ) Western blot analysis of phosphorylation using phos-tag in a recombinant OPN after kinase treatment. The unprocessed blot image is shown in . ( b ) Cell migration of H358 cells in the presence or absence of recombinant OPN pretreated with kinase and OPN function-blocking antibody (OPN Ab) either separately or in combinations. One-way ANOVA and Tukey post-hoc test, mean ± SEM of three independent assays conducted in triplicate. ( c ) Phos-tag ELISA analysis of phosphorylated OPN in MDA-MB435S CM after kinase treatment. Two-tailed unpaired Student’s t -test, mean ± SEM of three independent assays conducted in triplicate. ( d ) Cell migration of H358 cells in the presence or absence of the MDA-MB435S CM pretreated with kinase and OPN function-blocking antibody (OPN Ab), either separately or in combination. – and + indicate treatment without and with the indicated reagent. One-way ANOVA and Tukey post-hoc test, mean ± SEM of three independent assays conducted in triplicate.

Article Snippet: H358 (human lung cancer), H460 (human lung cancer), and MDA-MB435S (human melanoma) cell lines were obtained from ATCC.

Techniques: Migration, Western Blot, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Characterization of the MDA-MB-435S cell model. Surface expression of integrin subunit αV, integrin heterodimers αVβ3 and αVβ5, and integrin subunit β1 in melanoma cell line MDA-MB-435S and clones 2αV and 3αV obtained by stable transfection with integrin αV-specific shRNA. Cells were detached by EDTA and analyzed by flow cytometry upon incubation with antibodies against integrin subunit αV, β1, or integrin heterodimers αVβ3 or αVβ5 (black histogram), and isotype-matched antibody as a negative control (gray histogram), followed by rabbit FITC-conjugated-anti-mouse antibody (upper panel). Results from upper panel were presented as comparisons of MFIs within the cell model (lower panel). Representative data of three independent experiments yielding similar results are shown.

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: Characterization of the MDA-MB-435S cell model. Surface expression of integrin subunit αV, integrin heterodimers αVβ3 and αVβ5, and integrin subunit β1 in melanoma cell line MDA-MB-435S and clones 2αV and 3αV obtained by stable transfection with integrin αV-specific shRNA. Cells were detached by EDTA and analyzed by flow cytometry upon incubation with antibodies against integrin subunit αV, β1, or integrin heterodimers αVβ3 or αVβ5 (black histogram), and isotype-matched antibody as a negative control (gray histogram), followed by rabbit FITC-conjugated-anti-mouse antibody (upper panel). Results from upper panel were presented as comparisons of MFIs within the cell model (lower panel). Representative data of three independent experiments yielding similar results are shown.

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Expressing, Clone Assay, Stable Transfection, shRNA, Flow Cytometry, Incubation, Negative Control

Effect of integrin αV knockdown on cell sensitivity to antitumor drugs, PTX-induced apoptosis, cell proliferation, growth, spreading and migration. (A) Clones 2αV and 3αV demonstrate decreased sensitivity to cDDP and increased sensitivity to VCR and PTX as compared to parental MDA-MB-435S cells. Cells were seeded in 96-well plates and 24 h later treated with different concentrations of cDDP, VCR, and PTX. Cytotoxicity was measured by MTT assay. Results presented are representative of three independent experiments with similar results ± SD. Data were analyzed by unpaired Student’s t -test. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. (B) Clones 2αV and 3αV upon PTX treatment demonstrate increased apoptosis as compared to MDA-MB-435S cells. Cells were treated with 0.004 μg/mL of PTX for 72 h, harvested for Annexin V/PI staining and analyzed by flow cytometry to discriminate between live (lower left quadrant), apoptotic and/or necrotic cells (right quadrants). The representative data of three independent experiments yielding similar results are shown. (C) Cell proliferation in MDA-MB-435S cells and clones 2αV and 3αV. Cell proliferation was measured using ClickIT EdU assay. DNA synthesis was measured upon 2 h cell growth in medium supplemented with EdU and the amount of incorporated EdU was measured by flow cytometry. Comparison of average percentage of EdU + cells from three different experiments are shown. (D) Growth curve of MDA-MB-435S cells and clones 2αV and 3αV. Cells were seeded in 10 cm culture plates and counted on days 1, 2, and 3. The results presented are representative of three independent experiments with similar results. (E) Cell spreading of MDA-MB-435S cells and clones 2αV and 3αV. Live cell imaging was performed during 18–20 h upon seeding and cell spreading was compared using time lapse IRM images (30 min–12 h). Scale bar = 10 μm. (F) Decreased migration of clones 2αV and 3αV as compared to MDA-MB-435S cells. Serum starved (24 h) cells were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 μm. Averages of five microscope fields of three independently performed experiments ± SD are shown ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple Comparison. *** P < 0.001.

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: Effect of integrin αV knockdown on cell sensitivity to antitumor drugs, PTX-induced apoptosis, cell proliferation, growth, spreading and migration. (A) Clones 2αV and 3αV demonstrate decreased sensitivity to cDDP and increased sensitivity to VCR and PTX as compared to parental MDA-MB-435S cells. Cells were seeded in 96-well plates and 24 h later treated with different concentrations of cDDP, VCR, and PTX. Cytotoxicity was measured by MTT assay. Results presented are representative of three independent experiments with similar results ± SD. Data were analyzed by unpaired Student’s t -test. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. (B) Clones 2αV and 3αV upon PTX treatment demonstrate increased apoptosis as compared to MDA-MB-435S cells. Cells were treated with 0.004 μg/mL of PTX for 72 h, harvested for Annexin V/PI staining and analyzed by flow cytometry to discriminate between live (lower left quadrant), apoptotic and/or necrotic cells (right quadrants). The representative data of three independent experiments yielding similar results are shown. (C) Cell proliferation in MDA-MB-435S cells and clones 2αV and 3αV. Cell proliferation was measured using ClickIT EdU assay. DNA synthesis was measured upon 2 h cell growth in medium supplemented with EdU and the amount of incorporated EdU was measured by flow cytometry. Comparison of average percentage of EdU + cells from three different experiments are shown. (D) Growth curve of MDA-MB-435S cells and clones 2αV and 3αV. Cells were seeded in 10 cm culture plates and counted on days 1, 2, and 3. The results presented are representative of three independent experiments with similar results. (E) Cell spreading of MDA-MB-435S cells and clones 2αV and 3αV. Live cell imaging was performed during 18–20 h upon seeding and cell spreading was compared using time lapse IRM images (30 min–12 h). Scale bar = 10 μm. (F) Decreased migration of clones 2αV and 3αV as compared to MDA-MB-435S cells. Serum starved (24 h) cells were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 μm. Averages of five microscope fields of three independently performed experiments ± SD are shown ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple Comparison. *** P < 0.001.

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Migration, Clone Assay, MTT Assay, Staining, Flow Cytometry, EdU Assay, DNA Synthesis, Live Cell Imaging, Cell Culture, Microscopy

Clones 2αV and 3αV have decreased number and size of FAs and decreased amount of stress fibers per cell as compared to MDA-MB-435S cells. (A) Clones 2αV and 3αV show decreased expression of the integrin subunit αV and the FA marker, paxillin. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized and stained with antibody against integrin αV or paxillin followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed in all samples, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (B) Quantification of αV and paxillin, FA size and % of stress fibers per cell. Data presented as histograms or scatter plots (FA size) represent measurements of > 50 cells and are plotted as mean ± SD ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *** P < 0.001.

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: Clones 2αV and 3αV have decreased number and size of FAs and decreased amount of stress fibers per cell as compared to MDA-MB-435S cells. (A) Clones 2αV and 3αV show decreased expression of the integrin subunit αV and the FA marker, paxillin. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized and stained with antibody against integrin αV or paxillin followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed in all samples, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (B) Quantification of αV and paxillin, FA size and % of stress fibers per cell. Data presented as histograms or scatter plots (FA size) represent measurements of > 50 cells and are plotted as mean ± SD ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *** P < 0.001.

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Clone Assay, Expressing, Marker, Staining

Mass spectrometry analysis of IACs isolated from MDA-MB-435S cells and clones 2αV and 3αV. (A) Protein–protein interaction network of components identified by MS in IACs isolated form MDA-MB-435S cells. Shapes represent identified proteins and are labeled with gene symbols, arranged and colored according to their functional group as indicated (CMSC). In case of multiple functional terms assigned for each protein, the molecular function assigned by both databases has been chosen for interpretation of results. (B) Total identified IAC proteins in MDA-MB-435S cells (number of spectral counts ≥ 4, FDR < 5%, probability for protein identification ≥ 99.9%) were annotated using David GO database. To determine the sample enrichment, P -values related to GO terms of cellular components (GOTERM_CC_DIRECT), were used. Analysis of gene ontology terms was performed using REViGO tool. Statistically significant GO terms ( P = 0.05) were presented from highest P -value (bottom) to the lowest (top). (C) Volcano plot of MDA-MB-435S versus 2αV (red) and 3αV (black). To determine the significantly changed proteins −Log (FDR) ≥ 1 and fold change ≥ 1.5 were used. Upper left quadrant – proteins detected at lower level of spectra, upper right quadrant – proteins detected at higher level of spectra. (D) Venn diagram – proteins with higher (green arrow) and lower (red arrow) level of spectra detected in clones 2αV and 3αV versus MDA-MB-435S cells. Proteins found in both clones with changed abundances were showed in the intersected white area of the diagram. (E) DAVID GO analysis of proteins detected with higher (green arrow) and lower (red arrow) abundances. Statistically significant GO terms ( P = 0.05; dashed line) were presented from highest P -value (bottom) to the lowest (top).

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: Mass spectrometry analysis of IACs isolated from MDA-MB-435S cells and clones 2αV and 3αV. (A) Protein–protein interaction network of components identified by MS in IACs isolated form MDA-MB-435S cells. Shapes represent identified proteins and are labeled with gene symbols, arranged and colored according to their functional group as indicated (CMSC). In case of multiple functional terms assigned for each protein, the molecular function assigned by both databases has been chosen for interpretation of results. (B) Total identified IAC proteins in MDA-MB-435S cells (number of spectral counts ≥ 4, FDR < 5%, probability for protein identification ≥ 99.9%) were annotated using David GO database. To determine the sample enrichment, P -values related to GO terms of cellular components (GOTERM_CC_DIRECT), were used. Analysis of gene ontology terms was performed using REViGO tool. Statistically significant GO terms ( P = 0.05) were presented from highest P -value (bottom) to the lowest (top). (C) Volcano plot of MDA-MB-435S versus 2αV (red) and 3αV (black). To determine the significantly changed proteins −Log (FDR) ≥ 1 and fold change ≥ 1.5 were used. Upper left quadrant – proteins detected at lower level of spectra, upper right quadrant – proteins detected at higher level of spectra. (D) Venn diagram – proteins with higher (green arrow) and lower (red arrow) level of spectra detected in clones 2αV and 3αV versus MDA-MB-435S cells. Proteins found in both clones with changed abundances were showed in the intersected white area of the diagram. (E) DAVID GO analysis of proteins detected with higher (green arrow) and lower (red arrow) abundances. Statistically significant GO terms ( P = 0.05; dashed line) were presented from highest P -value (bottom) to the lowest (top).

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Mass Spectrometry, Isolation, Clone Assay, Labeling, Functional Assay

MS data validation in MDA-MB-435S cells and clones 2αV and 3αV. (A) Clones 2αV and 3αV show decreased expression of vinculin, talin 1/2 and α-actinin 1 as compared to MDA-MB-435S cells. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, incubated with antibodies against vinculin, talin 1/2 or α-actinin 1 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed in all samples, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (B) Quantification data of results in (A) presented as histograms (FA number) and scatter plots (FA or α-actinin 1 size) represent measurements of > 50 cells and are plotted as mean ± SD ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. * P < 0.05; ** P < 0.01; *** P < 0.001. (C) WB analysis of IAC proteins from clones 2αV and 3αV and MDA-MB-435S cells. Forty-eight hours after seeding, IACs were isolated and WB analysis was performed. The results presented are representative of three independent experiments yielding similar results.

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: MS data validation in MDA-MB-435S cells and clones 2αV and 3αV. (A) Clones 2αV and 3αV show decreased expression of vinculin, talin 1/2 and α-actinin 1 as compared to MDA-MB-435S cells. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, incubated with antibodies against vinculin, talin 1/2 or α-actinin 1 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed in all samples, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (B) Quantification data of results in (A) presented as histograms (FA number) and scatter plots (FA or α-actinin 1 size) represent measurements of > 50 cells and are plotted as mean ± SD ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. * P < 0.05; ** P < 0.01; *** P < 0.001. (C) WB analysis of IAC proteins from clones 2αV and 3αV and MDA-MB-435S cells. Forty-eight hours after seeding, IACs were isolated and WB analysis was performed. The results presented are representative of three independent experiments yielding similar results.

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Clone Assay, Expressing, Incubation, Staining, Isolation

Characterization of αVβ5-associated adhesion complexes. (A) The most abundant integrin subunits found in IACs. Dataset consists of at least two different experiments. Average spectral count number shown. (B) Clones with integrin subunit αV knockdown show decreased expression of integrin αVβ5. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, and stained with anti-αVβ5 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (C) Quantification data of results in (B) presented as histogram (FA number) and scatter plot (FA size) represent measurements of > 50 cells and are plotted as mean ± SD ( n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *** P < 0.001. (D) Identification of reticular adhesion structures in MDA-MB-435S cells and clone 2αV. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, and stained for anti-αVβ5 followed by Alexa-Fluor 488-conjugated antibody (green), and anti-vinculin followed by Alexa-Fluor 647-conjugated antibody (purple). F-actin staining (red) was performed, nuclei were stained with DAPI (blue) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm.

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: Characterization of αVβ5-associated adhesion complexes. (A) The most abundant integrin subunits found in IACs. Dataset consists of at least two different experiments. Average spectral count number shown. (B) Clones with integrin subunit αV knockdown show decreased expression of integrin αVβ5. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, and stained with anti-αVβ5 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). F-actin staining (red) was performed, and nuclei were stained with DAPI (blue). Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. (C) Quantification data of results in (B) presented as histogram (FA number) and scatter plot (FA size) represent measurements of > 50 cells and are plotted as mean ± SD ( n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *** P < 0.001. (D) Identification of reticular adhesion structures in MDA-MB-435S cells and clone 2αV. Forty-eight hours after seeding on coverslips, cells were fixed, permeabilized, and stained for anti-αVβ5 followed by Alexa-Fluor 488-conjugated antibody (green), and anti-vinculin followed by Alexa-Fluor 647-conjugated antibody (purple). F-actin staining (red) was performed, nuclei were stained with DAPI (blue) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm.

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Clone Assay, Expressing, Staining

KANK2 knockdown in MDA-MB-435S cells increases sensitivity to MT poisons and decreases migration mimicking integrin αV knockdown. (A) Average MS data of number of spectra specific for KANK2 in MDA-MB-435S cell model. Dataset consists of at least two different experiments. (B) KANK2 is present in lower amount in IACs of clones 2αV and 3αV. WB analysis of KANK2 in isolated IAC proteins from clones 2αV and 3αV and MDA-MB-435S cells. Forty-eight hours after seeding, IACs were isolated and WB analysis was performed. The results presented are representative of two independent experiments yielding similar results. (C) Knockdown of KANK2 in MDA-MB-435S cells. WB analysis of KANK2 from MDA-MB-435S cells transfected with either control (si(-)) or KANK2-specific siRNA (si(KANK2)). Forty-eight hours after transfection total cell lysates were collected and WB analysis was performed. The results presented are representative of two independent experiments yielding similar results. (D) MDA-MB-435S cells upon KANK2 knockdown demonstrate increased sensitivity to VCR and PTX as compared to MDA-MB-435S cells transfected with control siRNA. Twenty-four hours upon transfection, cells were seeded in 96-well plates and 24 h later treated with different concentrations of VCR and PTX. Cytotoxicity was measured by MTT assay. Results presented are representative of three independent experiments with similar results ± SD. Data were analyzed by unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. (E) KANK2 knockdown decreases migration in MDA-MB-435S cells. Serum starved (24 h) cells, transfected previously with either control or KANK2-specific siRNA were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 μm. Averages of five microscope fields of three independently performed experiments ± SD are shown ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple Comparison. *** P < 0.001. (F) KANK2 knockdown in MDA-MB-435S cells does not alter cell size or amount of stress fibers but slightly alters appearance of MTs. Forty-eight hours after transfection of MDA-MB-435S cells with KANK2-specific or control siRNA cells were fixed, permeabilized, and stained with anti-KANK2 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). The α-tubulin or F-actin staining (red) was performed, nuclei were stained with DAPI (blue) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. Quantification data of results are presented as histograms represent measurements of > 50 cells and are plotted as mean ± SD ( n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *** P < 0.001.

Journal: Frontiers in Cell and Developmental Biology

Article Title: KANK2 Links αVβ5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration

doi: 10.3389/fcell.2020.00125

Figure Lengend Snippet: KANK2 knockdown in MDA-MB-435S cells increases sensitivity to MT poisons and decreases migration mimicking integrin αV knockdown. (A) Average MS data of number of spectra specific for KANK2 in MDA-MB-435S cell model. Dataset consists of at least two different experiments. (B) KANK2 is present in lower amount in IACs of clones 2αV and 3αV. WB analysis of KANK2 in isolated IAC proteins from clones 2αV and 3αV and MDA-MB-435S cells. Forty-eight hours after seeding, IACs were isolated and WB analysis was performed. The results presented are representative of two independent experiments yielding similar results. (C) Knockdown of KANK2 in MDA-MB-435S cells. WB analysis of KANK2 from MDA-MB-435S cells transfected with either control (si(-)) or KANK2-specific siRNA (si(KANK2)). Forty-eight hours after transfection total cell lysates were collected and WB analysis was performed. The results presented are representative of two independent experiments yielding similar results. (D) MDA-MB-435S cells upon KANK2 knockdown demonstrate increased sensitivity to VCR and PTX as compared to MDA-MB-435S cells transfected with control siRNA. Twenty-four hours upon transfection, cells were seeded in 96-well plates and 24 h later treated with different concentrations of VCR and PTX. Cytotoxicity was measured by MTT assay. Results presented are representative of three independent experiments with similar results ± SD. Data were analyzed by unpaired Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001. (E) KANK2 knockdown decreases migration in MDA-MB-435S cells. Serum starved (24 h) cells, transfected previously with either control or KANK2-specific siRNA were seeded in Transwell cell culture inserts and left to migrate for 22 h toward serum. Cells on the underside of the inserts were stained with crystal violet, photographed, and counted. Scale bar = 100 μm. Averages of five microscope fields of three independently performed experiments ± SD are shown ( n = 3). Data were analyzed by one-way ANOVA with Dunnett’s multiple Comparison. *** P < 0.001. (F) KANK2 knockdown in MDA-MB-435S cells does not alter cell size or amount of stress fibers but slightly alters appearance of MTs. Forty-eight hours after transfection of MDA-MB-435S cells with KANK2-specific or control siRNA cells were fixed, permeabilized, and stained with anti-KANK2 antibody, followed by Alexa-Fluor 488-conjugated antibody (green). The α-tubulin or F-actin staining (red) was performed, nuclei were stained with DAPI (blue) and IRM images were taken. Analysis was performed using TCS SP8 Leica. Scale bar = 10 μm. Quantification data of results are presented as histograms represent measurements of > 50 cells and are plotted as mean ± SD ( n = 2). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison. *** P < 0.001.

Article Snippet: Human melanoma cell line MDA-MB-435S (a spindle-shaped variant of the parental MDA-MB-435) was obtained from the American Type Culture Collection (ATCC, United States).

Techniques: Migration, Clone Assay, Isolation, Transfection, MTT Assay, Cell Culture, Staining, Microscopy

Cellular WM853 and MDA-MB-435S clone characteristics. ( a ) SIRT2 expression in SCW3 and SSW30 clones of WM853 cells and SCM1 and SSM15 clones of MDA-MB-435S as evidenced by Western blotting. ( b ) The cytotoxic effects of dasatinib treatment (48 h) on melanoma SCW3 and SSW30 clones of the WM853 cell line, as evidenced using the neutral red assay, mean ± SD, ( n = 3, independent experiments) ( c ) The cytotoxic effects of dasatinib treatment (48 h) on melanoma SCM1 and SSM15 clones of the MDA-MB-435S cell line, as evidenced using the neutral red assay, mean ± SD, ( n = 3, independent experiments).

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: Cellular WM853 and MDA-MB-435S clone characteristics. ( a ) SIRT2 expression in SCW3 and SSW30 clones of WM853 cells and SCM1 and SSM15 clones of MDA-MB-435S as evidenced by Western blotting. ( b ) The cytotoxic effects of dasatinib treatment (48 h) on melanoma SCW3 and SSW30 clones of the WM853 cell line, as evidenced using the neutral red assay, mean ± SD, ( n = 3, independent experiments) ( c ) The cytotoxic effects of dasatinib treatment (48 h) on melanoma SCM1 and SSM15 clones of the MDA-MB-435S cell line, as evidenced using the neutral red assay, mean ± SD, ( n = 3, independent experiments).

Article Snippet: The human melanoma cell lines MDA-MB-435S (stage: metastatic) and A375 (stage: metastatic) were obtained from ATCC (Manassas, VA., USA) and maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (PAN-Biotech GmbH, Aidenbach, Germany) at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Expressing, Clone Assay, Western Blot, Neutral Red Assay

Selected category of genes (based on a literature search) with changes in expression in MDA-MB-435S cells with SIRT2 downregulation as determined using RNA-seq.

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: Selected category of genes (based on a literature search) with changes in expression in MDA-MB-435S cells with SIRT2 downregulation as determined using RNA-seq.

Techniques: Expressing

Treatment with dasatinib impairs the migration of melanoma cells based on the scratch assay. ( a ) Image from a single representative experiment performed using WM853 SCW3 and SSW30 clones. ( b ) Image from a single representative experiment performed using MDA-MB-435S SCM1 and SSM15 clones. Quantification of above results is shown in the . 10 × 10 magnification.

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: Treatment with dasatinib impairs the migration of melanoma cells based on the scratch assay. ( a ) Image from a single representative experiment performed using WM853 SCW3 and SSW30 clones. ( b ) Image from a single representative experiment performed using MDA-MB-435S SCM1 and SSM15 clones. Quantification of above results is shown in the . 10 × 10 magnification.

Techniques: Migration, Wound Healing Assay, Clone Assay

SIRT2 downregulation impairs the response of melanoma cells to EGFR and EPHA2 activators. The generated melanoma clones were treated with selected concentrations of EGF (EGFR activator) and ephrin A1 (EPHA2 activator) for 10 min and/or 30 min, respectively. Then, protein lysates were prepared and analyzed by Western blotting. ( a ) Experiments performed using WM853 SCW3 and SSW30 clones. ( b ) Experiments performed using MDA-MB-435S SCM1 and SSM15 clones.

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: SIRT2 downregulation impairs the response of melanoma cells to EGFR and EPHA2 activators. The generated melanoma clones were treated with selected concentrations of EGF (EGFR activator) and ephrin A1 (EPHA2 activator) for 10 min and/or 30 min, respectively. Then, protein lysates were prepared and analyzed by Western blotting. ( a ) Experiments performed using WM853 SCW3 and SSW30 clones. ( b ) Experiments performed using MDA-MB-435S SCM1 and SSM15 clones.

Techniques: Generated, Clone Assay, Western Blot

SIRT2 downregulation impairs the expression and phosphorylation of tyrosine kinase receptor-associated pathways. The generated melanoma clones were treated with selected concentrations of dasatinib for 1 h. Then, protein lysates were prepared and analyzed by Western blotting. ( b ) Experiments performed using WM853 SCW3 and SSW30 clones. ( a ) Experiments performed using MDA-MB-435S SCM1 and SSM15 clones.

Journal: Cancers

Article Title: SIRT2 Contributes to the Resistance of Melanoma Cells to the Multikinase Inhibitor Dasatinib

doi: 10.3390/cancers11050673

Figure Lengend Snippet: SIRT2 downregulation impairs the expression and phosphorylation of tyrosine kinase receptor-associated pathways. The generated melanoma clones were treated with selected concentrations of dasatinib for 1 h. Then, protein lysates were prepared and analyzed by Western blotting. ( b ) Experiments performed using WM853 SCW3 and SSW30 clones. ( a ) Experiments performed using MDA-MB-435S SCM1 and SSM15 clones.

Techniques: Expressing, Generated, Clone Assay, Western Blot

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