Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: A) and B) Development of T1D in the spontaneous (A) and cell transfer (B) models of WT and CD44-deficient mice was monitored by measuring blood glucose. In the transfer model, irradiated CD44-deficient and WT young (6–8 weeks-old) male recipients were respectively transplanted with splenocytes from CD44-deficient and WT diabetic females. Percentage of diabetes-free mice (showing <250 mg/dL blood glucose) was recorded versus time. Statistical analysis by Breslow; A , P < 0.05; B , P < 0.005. C) The invasion capacity of infiltrating cells derived from WT (n = 6) and CD44-deficient (n = 6) NOD females (spontaneous model) was measured as indicated in Materials and Methods . A total of 754 pancreatic islets in each mouse group were scored by an uninformed observer. The percentage of islets showing each one of the infiltrating scores was calculated for each mouse, and the average values are presented. Islets from CD44-deficient mice display higher percentage of infiltrating scores (2, 3 and 4) than islets from wild type mice. P < 0.0001 by Pearson's χ2 test comparing the distribution of scores in the two mouse groups. D) Wild type or CD44-deficient pre-diabetic spleen cells were added to the top compartment of transwell migration chamber, separated by HA-coated filter (10 μg/filter) from the bottom compartment, and the % of cells migrating through the filter toward the chemoatractant in the bottom compartment was calculated by flow cytometry. Statistical analysis by Student’s- t- test. Accumulated data of 4 experiments (n = 8; error bars, SEM).

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: Irradiation, Derivative Assay, Migration, Flow Cytometry

Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: A) CD44-deficient non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44 +/+ →CD44 -/- ; marked #1) or CD44-deficient (CD44 -/- →CD44 -/- ; marked #2) diabetic NOD female donors. In addition, WT non-diabetic NOD male recipients were reconstituted with inflammatory spleen cells derived from WT (CD44 +/+ →CD44 +/+ ; marked #3) or CD44-deficient (CD44 -/- →CD44 +/+ ; marked #4) diabetic NOD female donors. Inflammatory cells are marked by arrows and local islet cells by arrowhead. The pancreases of all 4 combinations were removed after detection of diabetes or 200 days after cell transfer. Doing so, pancreases that were harvested approximately at the same time, were stained with anti-CD44 mAb. B) The % of diabetes-free mice (exhibiting less than 250 mg/dL urine glucose) was determined in corresponding four mouse groups, described in A . Statistical analysis by Breslow: #2 versus #3, P < 0.01; #1 versus #2 (follow up 75 to 200 days), P < 0.03; #3 versus #4 (follow up 20 to 75 days), P < 0.03.

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: Derivative Assay, Staining

Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: A) Enhanced caspase-3 activity is detected in cell extracts of WT than in those of CD44-deficient pancreatic islet cells. Pancreatic islet cells were freshly removed from WT and CD44-deficient pre-diabetic NOD females. Cell extracts from islets were subjected to Western blot analysis, using anti-cleaved caspase-3 antibody. A representative Fig of three experiments. (B and C) Pancreata were harvested from 10-weeks-old normoglycemic WT and CD44-deficient NOD females (n = 5 in each group). β-cell apoptosis was assessed using TUNEL assay (green) and co-staining for insulin (red) and DNA (blue). B) Apoptosis was calculated as percent of TUNEL positive nuclei (+/- SEM) in insulin-positive cells out of the total number of insulin-positive cells per mouse. WT (white bar, ~17,560 total β-cells); CD44-deficient (black bar, ~14,800 total β-cells). C) Pictures are representative images of islets from WT (left panel) and CD44-deficient NOD females (right panel). Original images were taken at a magnification of x40. Inset images were digitally increased x5. White arrowheads point to apoptotic β-cells.

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: Activity Assay, Western Blot, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

doi: 10.1371/journal.pone.0143589

Figure Lengend Snippet: (A and B) In vitro pro-inflammatory conditions. A) Griess assay. Wild type and CD44-deficient pancreatic islet cells were incubated with medium (-) or Cytomix for 24 and 48 h. Nitrite release was assessed by Griess assay (n = 3). Inset: Cell extracts from pancreatic islets described in ( A ) were subjected to Western blot analysis, using anti-iNOS antibody. A representative Fig of three experiments. B) Glucose-stimulated insulin secretion (GSIS). Pancreatic islet cells from WT and CD44-deficient NOD mice were incubated with medium (-) or Cytomix for 48 h and then stimulated with 3.3 or 16.7 mM glucose for additional one hour and their ability to secrete insulin was measured by ELISA. Inset: Insulin content in each one of the samples (n = 4–5) (error bars, SEM). Statistical analysis for A and B by standard two-tailed Student’s t -test. A , one representative experiment of five. B , one representative experiment of two. (C and D) Ex vivo pro-inflammatory conditions. C) Freshly isolated pancreatic islet cells from WT and CD44-deficient pre-diabetic NOD females were processed as described in ( B ) (n = 6–8) (error bars, SEM). D) Content of insulin in pancreatic islets of WT and CD44-deficient pre-diabetic NOD females was analyzed by ELISA. Statistical analysis in C and D by standard two-tailed Student’s t -test. The statistical analysis in C shows that the ex vivo insulin release of CD44-deficient pancreatic islets at 12 and 14 weeks is significantly higher (p<0.05) than the insulin release of the corresponding WT cells.The statistical analysis in D shows that the insulin content in pancreatic islets of 14 wks-old pre-diabetic WT mice is significantly different ( P < 0.05) from all other samples. (n = 6–8). In C—D , at least one representative experiment of two.

Article Snippet: CD44-deficient DBA/1 mice were generated by backcrossing CD44-deficient C57BL/6 mice into DBA/1 mice (Harlan, Jerusalem, Israel) for 8 generations [ ].

Techniques: In Vitro, Griess Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Ex Vivo, Isolation

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Schematic representation of CD44S. A : The extracellular (blue), transmembrane (red), and cytoplasmic (green) domains are illustrated for standard CD44S. The extracellular domain is characterized by cysteine residues that form disulfide bonds (maroon beads), including the two signature disulfide bonds that form the backbone of the link domain. Several extracellular putative phosphorylation sites as well as docking sites for MMP-9 and cleavages of CD44 by MT1-MMP (in conjunction with ADAMs 10 and 17) in the CD44 stem region and the transmembrane region by γ-secretase are shown. The cytoplasmic domain is characterized by three cytoplasmic phosphorylation sites (purple arrows) that modulate CD44 ezrin linking [ , ]. Note the extracellular β amyloid-like fragment and the nuclear and mitochondrial trafficking signals. Abbreviations for putative phosphorylation sites are: PKA, protein kinase A; PKC, protein kinase C; CKII, casein kinase II; SRC, sarcoma tyrosine kinase; INSR, insulin receptor kinase; ERM, ezrin, radixin, moesin. B : Gene structure of canonical CD44 with invariant exons 1–5, variable spliced exons 6–14, invariant exons 15–16 forming stem region, transmembrane exon 17, and cytoplasmic domain exon 19 is illustrated. Note exon 18 is spliced out . Adenoviral constructs were the standard (Ad-CD44S) and truncated soluble (Ad-sCD44) isoforms; the isolated sCD44 is depicted for comparison to the constructs.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques: Construct, Isolation

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Effects of Ad-sCD44 and Ad-CD44S transduction on CD44 levels on day 7 in human trabecular meshwork cells. CD44 concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and are expressed in ng/ml in both the adenoviral media and cell lysates. Fetal bovine serum (FBS) was used as negative (Neg) control; human serum (HS) was used as a positive (Pos) control. Data are the mean ± SEM; n=4. Analysis of variance (ANOVA) was used to determine significance between: Ad-Empty media versus Ad-sCD44 media ** p<0.01; Ad-Empty lysate versus Ad-sCD44 lysate, †† p<0.01; Ad-CD44S media versus Ad-sCD44 media, ‡‡ p<0.01; Ad-CD44S lysate versus Ad-sCD44 lysate, ‡‡‡ p<0.001; Ad-Empty lysate versus Ad-CD44S lysate, §§§ p<0.001.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques: Transduction, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Effects of Ad-sCD44 and Ad-CD44S transduction on CD44 levels on day 7 in human trabecular meshwork cells. CD44 was determined by western blots of culture media and lysates of trabecular meshwork cells. The lane numbers signify the following: Lane 1, Ad-sCD44 media; lane 2, Ad-sCD44 cell lysate; lane 3, Ad-CD44s media; lane 4, Ad-CD44s cell lysate; lane5, Ad-empty media; lane 6 Ad-empty cell lysate; lane 7, fetal bovine serum (FBS) control; and lane 8, isolated 32-kDa sCD44. Western blots were performed in triplicate.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques: Transduction, Western Blot, Isolation

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Enzyme-linked immunosorbent assay determination on day 14 of aqueous CD44 concentration in Ad-CD44S and Ad-sCD44 transduced mice eyes. A distribution plot of sCD44 concentration in the mouse aqueous humor is displayed for eyes treated with Ad-CD44S (n=7) or Ad-sCD44 (n=9) adenoviruses and their respective uninjected contralateral eyes. Data are expressed as mean ± SEM, **p<0.01, compared with the no-injection group using one-way analysis of variance (ANOVA) followed by Dunnett’s test.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Injection

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Effects of Ad-CD44S and Ad-sCD44 transduction on mouse intraocular pressure. One eye of BALB/cJ mice was injected intravitreally with 6×10 7 pfu with Ad-CD44S or Ad-sCD44 at day 0. The contralateral eye of mice was uninjected and served as a control. Results are expressed as mean ± SEM (n=8 for the injected groups, n=16 for control). The statistical results are: *p<0.05, **p<0.01, *** p<0.001 compared with the no-injection group using one-way analysis of variance (ANOVA) followed by Dunnett’s test.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques: Transduction, Injection

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Effects of CD44 neutralizing antibody on sCD44 normalized flow rate. Porcine anterior segments were perfused with 1 ng/ml sCD44 (as shown in ), 1 ng/ml sCD44 + 0.05 ng/ml CD44 antibody, 1 ng/ml CD44 + 0.5 ng/ml CD44 antibody, and 1 ng/ml CD44 + 5.0 ng/ml CD44 antibody. Time points are the mean ± SEM and the number of experiments are shown in the insert. sCD44 versus sCD44 + 0.05 ng/ml CD44 antibody. The statistical comparisons are: sCD44 versus sCD44 + 0.05 ng/ml CD44 antibody, *p<0.05, **p<0.01; 0.05 ng/ml CD44 antibody versus 5.0 ng/ml CD44 antibody, † p<0.05.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques:

Journal: Molecular Vision

Article Title: sCD44 overexpression increases intraocular pressure and aqueous outflow resistance

doi:

Figure Lengend Snippet: Effects of γ-secretase inhibitor (GSI) on sCD44 normalized flow rate in perfusion cultured porcine anterior segments. Porcine anterior segments were perfused at constant pressure with 1 ng/ml sCD44 (as shown in ), 1 ng/ml CD44 + 15 mM GSI and 15 mM GSI alone. Time points are the mean ± SEM and the number of experiments are shown in the insert. The statistical comparisons are: sCD44 versus GSI alone, *p<0.05; sCD44vsCD44 +15mM GSI, † p<0.05, †† p<0.01. DMEM is Dulbecco’s Modified Eagle’s Medium.

Article Snippet: Standard CD44 (CD44S) wild-type human CD44v4 cDNA ( NM_001001391 ) was obtained from Origene (Rockville, MD) in the pCMV6-XL5 vector. sCD44 cDNA was produced by introducing a stop codon after exon 5 ( ).

Techniques: Cell Culture, Modification

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

Journal: Cytotechnology

Article Title: Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro

doi: 10.1007/s10616-015-9857-x

Figure Lengend Snippet: Represents the list of antibodies used to characterize the HSKs and DMSCs

Article Snippet: CD-44 , Anti-human CD44 , 1:50 , BD Pharmingen (550989) , Immunophenotyping by flow cytometry.

Techniques: Transduction, Cell Culture, In Situ, Staining, Flow Cytometry, Immunohistochemistry, Immunofluorescence, Western Blot

Journal: Cytotechnology

Article Title: Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro

doi: 10.1007/s10616-015-9857-x

Figure Lengend Snippet: Immunophenotyping of the HEKs: flow cytometry revealed the positive expression of these ketatinocyte markers such as (a, b) E-cadherin along with its specific isotype control and (c, d) keratin-5 with isotype control. Expansion and characterization of dermal-derived mesenchymal-like stromal cells (DMSCs): e Phase contrast photographs of DMSCs at early passage (P5); f late passages (P15), respectively. Immunofluorescence analysis of DMSCs using: g–i anti-vimentin; j–l anti-CD44; m–o anti-CD73 antibodies. Vimentin being a cytoskeletal protein shows cytoskeletal localization while CD44 and CD73 being cell surface antigens demonstrate surface localization. DAPI was used as a nuclear stain. Scale bars 100 μm (e–o). (Color figure online)

Article Snippet: CD-44 , Anti-human CD44 , 1:50 , BD Pharmingen (550989) , Immunophenotyping by flow cytometry.

Techniques: Flow Cytometry, Expressing, Derivative Assay, Immunofluorescence, Staining