gfp-tagged wild-type cd44 plasmids #137823  (Addgene inc)

Journal: Breast Cancer Research : BCR

Article Title: A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

doi: 10.1186/bcr3614

Figure Lengend Snippet: CD44 affiliation with lipid rafts is reduced during migration of highly-invasive breast cancer cells. Sucrose density gradient fractionation was used to isolate lipid rafts from nonmigrating (a confluent cell monolayer) versus migrating (2 hours post scratch-wounding) MDA-MB-231 cells. (A) Raft fractions were identified on the basis of peak biochemical activity of the lipid raft-affiliated enzyme alkaline phosphatase. (B) Enrichment of the marker proteins Flotillin-1 (Flot-1) and transferrin receptor (TfR) by immuno-dot blot was further used to define the identity of respectively raft versus nonraft fractions. (C) The western blot expression profile of CD44 and its binding partners in MCF-10a normal-like breast cells revealed increased recovery of raft-affiliated CD44 after 2 hours of migration compared with nonmigrating controls. This was verified by calculation of the raft affiliation ratio from the quantification of three independent experiments (histogram). The CD44 binding partners tested were exclusively recovered from nonraft fractions. (D) CD44 recovery from lipid raft fractions of highly-invasive MDA-MB-231 breast cancer cells was reduced in migrating relative to nonmigrating conditions; and was paralleled by increased CD44 recovery from nonraft fractions in migrating conditions. This observation was verified by calculation of the raft affiliation ratio from the quantification of three independent experiments (histogram). The CD44 binding partners tested were exclusively recovered from nonraft fractions. Error bars, standard error of the mean; n = 3 experiments. *P < 0.05, Student’s t test . NR, nonlipid raft; OD, optical density.

Article Snippet: A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration.

Techniques: Migration, Fractionation, Activity Assay, Marker, Dot Blot, Western Blot, Expressing, Binding Assay

Journal: Breast Cancer Research : BCR

Article Title: A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

doi: 10.1186/bcr3614

Figure Lengend Snippet: CD44 palmitoylation-impaired constructs have reduced affiliation with lipid rafts. (A) Schematic representation of palmitoylation-impaired CD44 constructs generated by site-directed mutagenesis of human CD44. Single-site mutants were termed C286A and C286S, while double-site mutants (C286S + C295A, C286A + C295A) were termed SA and AA respectively. (B) MDA-MB-231 cells were transfected with full-length CD44 (CD44WT) or CD44 single-site (C286A, C286S) and double-site (SA, AA) palmitoylation-impaired mutants and were selected for 48 hours. Whole cell lysates revealed increased expression of CD44 in the transfected cells. (C) Full-scale lipid raft extractions confirmed reductions in raft-affiliated CD44 in cells expressing the mutant constructs relative to controls. (D) Calculation of the lipid raft:nonlipid raft (LR:NR) ratio for multiple experiments confirmed statistically significant reductions in CD44 raft affiliation upon expression of the CD44 palmitoylation-impaired mutants. (E) Triton X-100-insoluble and Triton X-100-soluble fractions were isolated and confirmed to be enriched in respectively lipid raft (Flotillin-1 (Flot-1)) or nonraft (transferrin receptor (TfR)) markers. CD44 recovery from raft-enriched fractions was reduced in cells overexpressing mutant CD44, and paralleled by increased recovery of CD44 in nonraft fractions. (F) Calculation of the affiliation ratio of CD44 with detergent-insoluble versus detergent-soluble fractions confirmed reductions in raft-affiliated CD44 from cells expressing mutant constructs relative to controls. Error bars, standard error of the mean; n = 3. # P < 0.05 (C286S vs. control); *P < 0.05; **P < 0.01, Student’s t test . HAM, hydroxylamine; WT, wild-type.

Article Snippet: A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration.

Techniques: Construct, Generated, Mutagenesis, Transfection, Expressing, Isolation

Journal: Breast Cancer Research : BCR

Article Title: A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

doi: 10.1186/bcr3614

Figure Lengend Snippet: Reduced CD44 palmitoylation is paralleled by increased cell migration. (A) Biotin-1-biotinamido-4-(4′-(maleimidomethyl)cyclohexanecarboxamido)butane (biotin-BMCC) assays were used to measure palmitoylated CD44 (CD44-Palm) in cells expressing representative single and double palmitoylation mutants, and were compared with total CD44 levels (CD44-Total). Isotype-matched IgG was used as a negative control for CD44 immunoprecipitations (IPs), and omission of hydroxylamine (HAM) reagent was a BMCC-negative control. CD44-Palm was reduced in cells overexpressing CD44 palmitoylation-impaired mutant constructs. (B) Densitometric quantification of multiple experiments confirmed significant reductions in CD44-Palm relative to CD44-Total in mutant cells. Error bars, standard error of the mean (SEM); n = 3. *P < 0.05; **P < 0.01, Student’s t test . (C) Phase contrast micrographs of scratch-wound migration assays performed in MDA-MB-231 cells transfected for 48 hours with CD44WT or CD44 single-site (C286A, C286S) or double-site (SA, AA) palmitoylation-impaired mutants. The graph was constructed by expressing wound width measurements at each time point relative to its cognate time = 0 value. Cell migration was significantly enhanced in mutant-expressing cells compared with control cells, as indicated in the graphical representation of multiple experiments. Error bars, SEM; n = 3. # φ* P < 0.05; **P < 0.01, two-way analysis of variance . WT, wild-type.

Article Snippet: A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration.

Techniques: Migration, Expressing, Negative Control, Mutagenesis, Construct, Transfection

Journal: Breast Cancer Research : BCR

Article Title: A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

doi: 10.1186/bcr3614

Figure Lengend Snippet: Reducing CD44 is sufficient to alter normal phenotype in breast cells. MCF-10a cells were transfected for 48 hours with full-length CD44 (CD44WT), and single-site (C286A, C286S) or double-site (SA, AA) palmitoylation-impaired mutants. (A) Triton X-100-insoluble and Triton X-100-soluble fractions were isolated, and were confirmed to be enriched in respectively lipid raft (Flotillin-1 (Flot-1)) and nonraft (transferrin receptor (TfR)) marker proteins. Overexpression of mutant CD44 reduced CD44 recovery from raft-containing fractions compared with that in untransfected controls and CD44WT-expressing cells. (B) Calculated ratios of CD44 affiliation with detergent-insoluble fractions reflected significant reductions in CD44 raft affiliation in mutant cells compared with untransfected control cells, and furthermore in CD44 double-site mutants compared with CD44WT. (C) CD44 palmitoylation was assessed by 1-biotinamido-4-(4′-(maleimidomethyl)cyclohexanecarboxamido)butane (BMCC) assay in CD44 immunoprecipitates of control, CD44WT-expressing and CD44 mutant-expressing cells. Palmitoylated CD44 was detected with streptavidin (CD44-Palm), and total CD44 detected using CD44 antibody (CD44-Total). No CD44 was immunoprecipitated in the isotype-matched IgG control lanes, and no palmitoylated CD44 was detected in the hydroxylamine (HAM)-negative control conditions. In the HAM-positive condition, palmitoylated CD44 was reduced in mutant-expressing cells. (D) Quantification of palmitoylated CD44 (as a ratio of total CD44) revealed significant reductions in cells overexpressing a double-site CD44 palmitoylation-impaired mutant (AA) relative to control or CD44WT-expressing cells ( *P < 0.05; **P < 0.01, Student’s t test). (E) Phase-contrast micrographs 48 hours post transfection revealed a change in the morphology of control MCF-10a colonies (Ctrl) following CD44 overexpression, with a protrusive, disseminated epithelial-to-mesenchymal transition-like appearance in all of the CD44-overexpressing cells (arrows), which was particularly pronounced in those expressing the CD44 palmitoylation-impaired mutants. (F) Migration was measured via scratch-wound assays, in which wound widths at each time point were expressed relative to their cognate measurement at time = 0 for each condition. These assays revealed significant enhancements in the migratory capacity of MCF-10a cells expressing CD44 palmitoylation-impaired mutants relative to control cells ( *P < 0.05, two-way analysis of variance ). Error bars, standard error of the mean; n = 3 experiments. WT, wild-type.

Article Snippet: A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration.

Techniques: Transfection, Isolation, Marker, Over Expression, Mutagenesis, Expressing, Immunoprecipitation, Negative Control, Migration

Journal: Breast Cancer Research : BCR

Article Title: A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

doi: 10.1186/bcr3614

Figure Lengend Snippet: Decreased CD44 palmitoylation is paralleled by increased co-association with ezrin. (A) CD44 immunoprecipitates from different MDA-MB-231 migration time points were probed for palmitoylated CD44 by 1-biotinamido-4-(4′-(maleimidomethyl)cyclohexanecarboxamido)butane (BMCC) assay. Palmitoylated CD44 was detected using streptavidin (CD44-Palm), and total CD44 detected using a CD44 primary antibody (CD44-Total). The absence of hydroxylamine (HAM) treatment was a negative control for palmitoylated CD44. CD44-Palm levels decreased over the migration time course. (B) Quantification of CD44-Palm as a ratio of CD44-Total revealed significant time-dependent reductions during migration. Error bars, standard error of the mean; n = 3 experiments. *P < 0.05, Student’s t test. (C) CD44 was immunoprecipitated from whole cell lysates of MDA-MB-231 cells at the indicated migration time points, with IgG as an internal control. Immunoprecipitates were probed for CD44 and ezrin, with the IgG heavy chain band intensity considered as a loading control. Ezrin-CD44 co-immunoprecipitation (IP) was increased after 1 and 2 hours of cell migration compared with nonmigrating cells (time = 0). (D) CD44 was immunoprecipitated from migrating (2 hours) untransfected MDA-MB-231 (Ctrl), WT-expressing, single and double palmitoylation mutant-expressing cells. The latter cells had a notable increase in CD44-ezrin co-association. IP experiments are representative of three independent experiments. (E) Confluent breast primary cells were stained for CD44 (green) and Flotillin-1 (Flot-1, red). Nuclei were stained with DAPI and are shown in grey. Ductal carcinoma in situ (DCIS) primary cells demonstrated the highest co-localisation of the two proteins compared with invasive ductal carcinomas (IDC). (F) Whole cell lysates of primary cultures from two nontumour (NT), and six IDC of tumour grade 2 and 3 (three of each) were subjected to BMCC assays to compare palmitoylated CD44 levels. Most palmitoylated CD44 (CD44-Palm) was recovered from NT cultures, while all patient IDC cultures displayed comparatively little CD44-Palm. Palmitoylated CD44 levels densitometrically normalised to total CD44 (CD44-Total) further demonstrated increased levels of CD44-Palm in NT samples . Error bars, standard deviation of duplicates in one experiment .

Article Snippet: A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration.

Techniques: Migration, Negative Control, Immunoprecipitation, Expressing, Mutagenesis, Staining, In Situ, Standard Deviation

Journal: Breast Cancer Research : BCR

Article Title: A novel mechanism of regulating breast cancer cell migration via palmitoylation-dependent alterations in the lipid raft affiliation of CD44

doi: 10.1186/bcr3614

Figure Lengend Snippet: Schematic representation of the proposed model of regulation of breast cancer cell migration via CD44 localisation in lipid rafts. When CD44 is affiliated with lipid rafts via palmitoylation of its cysteine residues, it is sequestered from binding its cytoplasmic binding partners and thus migration is restrained. However, when CD44 translocates outside lipid rafts in its de-palmitoylated state, its cytoplasmic tail is free to bind its cytoskeletal partners, subsequently facilitating cell migration. ERM, Ezrin/Radixin/Moesin.

Article Snippet: A pOTB7 plasmid encoding wild-type human CD44 (imaGenes; Source Bioscience) was grown on Luria-Bertani agar plates with 20 μg/ml chloramphenicol and subsequently in Luria-Bertani broth with the same antibiotic concentration.

Techniques: Migration, Binding Assay

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Tumor-promoting effects by the overexpression of Eno1, Eef2, and VCL in 4T1.2 mammary tumor cells, and tumor-suppressing effects by the administration of their recombinant proteins. The double asterisk indicates p < 0.01. CN = control, Eno1 = enolase 1, VCL = vinculin, and Hsp = Hsp90ab1. ( A-C ) Elevation in EdU-based proliferation, transwell invasion, and the upregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail by the overexpression of Eno1, Eef2, and VCL in 4T1.2 tumor cells. ( D ) Decrease in EdU-based proliferation by the administration of Eno1, Eef2, and VCL recombinant proteins. ( E ) Reduction in transwell invasion by the administration of Eno1, Eef2, and VCL recombinant proteins. ( F ) Downregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail in 4T1.2 tumor cells by the administration of Eno1, Eef2, and VCL recombinant proteins. ( G ) Co-immunoprecipitation of CD44 by Eno1 in 4T1.2 cells. ( H-I ) Suppression of the reduction in MTT-based viability by Eno1 in response to the silencing of CD44. ( J ) Downregulation of Lrp5, Runx2, MMP9 and Snail in 4T1.2 cells by the administration of Eno1 recombinant proteins, and the suppression by the silencing of CD44. ( K-L ) Western blotting of wild-type and mutant CD44 proteins, which were pulled down by Halo-tagged Eno1 proteins. PD = pull-down assay, NC = negative control, pl = Eno1 or CD44 proteins from plasmid transfection, MT = mutant CD44 without a cytoplasmic domain, and WT = wild-type CD44.

Article Snippet: The protein lysates were prepared from 4T1.2 cells, which were transfected with GFP-tagged wild-type CD44 plasmids (#137823, Addgene), GFP-tagged mutant CD44 plasmids lacking the cytoplasmic domain (#137822, Addgene), or Halo-tagged Eno1 plasmids (#175330, Addgene).

Techniques: Over Expression, Recombinant, Control, Immunoprecipitation, Western Blot, Mutagenesis, Pull Down Assay, Negative Control, Plasmid Preparation, Transfection