Journal: International Journal of Molecular Sciences

Article Title: Synergy between Readthrough and Nonsense Mediated Decay Inhibition in a Murine Model of Cystic Fibrosis Nonsense Mutations

doi: 10.3390/ijms22010344

Figure Lengend Snippet: Readthrough with G418 in a cell based reporter system and G542X intestinal organoids ( A ). Firefly luciferase activity in 3T3 cells transfected with pFluc190UGA and treated with the indicated dose of G418. n = 3 wells per dose. * p < 0.0001 by one way ANOVA with post hoc Tukey test, ± SD. ( B ). Representative brightfield images of G542X intestinal organoids with indicated treatments at 0 and 180 min following stimulation with 10 µM forskolin. Scale bar is 100 µm. ( C ). FIS curves of intestinal organoids images every 15 min following forskolin stimulation. n = 3 wells per treatment group. ( D ). AUC measurements recorded from 1C. **** p < 0.0001, ^ p < 0.0001 vs. DMSO by one-way ANOVA with post hoc Tukey test.

Article Snippet: The following day, the cells were transfected with 100 ng per well of pFluc190UGA (a kind gift from James Inglese; Addgene plasmid #41046) [ ] using Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA, USA, Cat. #11668030).

Techniques: Luciferase, Activity Assay, Transfection

Journal: Molecular Cancer

Article Title: Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

doi: 10.1186/s12943-014-0279-8

Figure Lengend Snippet: Syndecan-2 is a regulator of MDA-MB231 cytoskeleton and behaviour. (A) Specific knockdown of mRNA levels after syndecan-1, −2 or −4 siRNA treatments. mRNA was extracted from cells 48 h after transfection with syndecan-1, syndecan-2, syndecan-4 or control siRNAs. Levels of other syndecan mRNAs were not affected by syndecan-2 knockdown (left). (B) Flow cytometry analysis confirmed loss of cell surface syndecan-2 after silencing syndecan-2 with siRNA. (C) Syndecan-2 depleted cells were stained for F-actin, which showed extensive microfilament bundle formation. Bar = 25 μm. (D) Spread cell areas were increased by syndecan-2 depletion, n ≥ 50 cells per condition. (E) Cadherin-11 containing adherens junctions were visible after syndecan-2 depletion. Bar = 25 μm. (F, G) Type I collagen invasion (F) and degradation (G) were reduced in syndecan-2 depleted cells. Higher magnification images are shown on the lower panels of G. Bar = 100 μm. (H) Cells depleted of each syndecan individually or in combination, by siRNA treatment were stained for focal adhesions (arrows) with a paxillin antibody. Quantification of focal adhesion (FA) size and number was done by one-way ANOVA with Tukey’s post-hoc test. Bar = 25 μm. Error bars = s.e.m. from three independent experiments. ***p < 0.001, n.s.; not significant by two-tailed paired t-test.

Article Snippet: Cells were transfected with siRNA targeting syndecan-1 (sc-36587, Santa Cruz Biotechnology, CA, USA), syndecan-2 (sc-41045, Santa Cruz Biotechnology, CA, USA), syndecan-4 (5′-ggccgauacuucuccggaguu-3′, Qiagen, Frederick, MD, USA), MMP14 (5′-caggcaaagcugaugcagauu-3′, Qiagen), PAR-1 (5′-aaggcuacuaugccuacuacuuu-3′, Thermo Fisher Scientific, Waltham, MA, USA), caveolin-1 (siGENOME SMARTpool, Thermo Scientific), caveolin-2 (siGENOME SMARTpool, Thermo Scientific) or non-targeting siRNA (siGENOME SMARTpool, Thermo Scientific) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions.

Techniques: Transfection, Flow Cytometry, Staining, Two Tailed Test

Journal: Molecular Cancer

Article Title: Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

doi: 10.1186/s12943-014-0279-8

Figure Lengend Snippet: Heparan sulfate treatment causes loss of syndecan-2 and gain of syndecan-4 on the cell surface. MDA-MB231 cells were analysed at 2, 16 and 24 h after treatment with 20 μg/ml heparan sulfate for cell surface levels of syndecans-1, −2 and −4. Control cells were untreated. FACS analysis showed decreased syndecan-2 to near background, while levels of syndecan-4 increased. Syndecan-1 levels were unchanged throughout the treatment.

Article Snippet: Cells were transfected with siRNA targeting syndecan-1 (sc-36587, Santa Cruz Biotechnology, CA, USA), syndecan-2 (sc-41045, Santa Cruz Biotechnology, CA, USA), syndecan-4 (5′-ggccgauacuucuccggaguu-3′, Qiagen, Frederick, MD, USA), MMP14 (5′-caggcaaagcugaugcagauu-3′, Qiagen), PAR-1 (5′-aaggcuacuaugccuacuacuuu-3′, Thermo Fisher Scientific, Waltham, MA, USA), caveolin-1 (siGENOME SMARTpool, Thermo Scientific), caveolin-2 (siGENOME SMARTpool, Thermo Scientific) or non-targeting siRNA (siGENOME SMARTpool, Thermo Scientific) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions.

Techniques:

Journal: Molecular Cancer

Article Title: Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

doi: 10.1186/s12943-014-0279-8

Figure Lengend Snippet: Caveolin-2 interacts with syndecan-2 and regulates cell adhesion in MDA-MB231 cells. (A) Syndecan-2, but not syndecan-4, was co-immunoprecipitated with caveolin-2 from cell lysates. Co-immunoprecipitation was not affected by ectopic glycosaminoglycan treatment of the cells (UT; untreated, CS; chondroitin sulfate, HS; heparan sulfate). (B) Confocal laser scanning microscopy and profile of the line scanning (white arrow on image) confirmed partial co-localization of syndecan-2 (green) and caveolin-2 (red). (C) Caveolin-2 levels were reduced where syndecan-2 was depleted by siRNA, compared with control siRNA. (D) Western blotting verified the knockdown efficiency of siRNA targeting caveolin-1 and −2 compared to control siRNA. Downregulation of caveolin-1 reduced the expression of caveolin-2 by around 30%, but knockdown of caveolin-2 had no impact on caveolin-1 levels. (E) F-actin containing microfilament bundles were abundant after caveolin-2, but not caveolin-1 depletion. Bar = 25 μm. (F) Spread cell areas were measured in caveolin-2 depleted cells and control cells, n ≥ 50 per condition. (G) Adherens junctions and focal adhesions (arrows) were characteristic of caveolin-2 depleted cells, shown by cadherin-11 and p120-catenin, or paxillin distributions. Bar = 25 μm. (H) Microfilament bundles formed in response to either syndecan-2 or caveolin-2 knockdown were sensitive to 30 μM Rho kinase inhibitor, Y-27632. Bar = 25 μm. (I) Diphosphorylated myosin light chain (ppMLC; Thr18/Ser19) was enhanced in both syndecan-2 and caveolin-2 depleted cells. Densitometry analysis of western blots for ppMLC and total MLC were normalised to β-tubulin. Similar results were obtained in three independent experiments. (J, K) Caveolin-2 depleted cells had reduced ability to invade or degrade type I collagen gels. Higher magnification images are shown in the lower panels. Caveolin-1 depletion also reduced collagen gel invasion. Bar = 100 μm. Error bars = s.e.m. **p < 0.01, ***p < 0.001, n.s.; not significant by two-tailed paired t-test.

Article Snippet: Cells were transfected with siRNA targeting syndecan-1 (sc-36587, Santa Cruz Biotechnology, CA, USA), syndecan-2 (sc-41045, Santa Cruz Biotechnology, CA, USA), syndecan-4 (5′-ggccgauacuucuccggaguu-3′, Qiagen, Frederick, MD, USA), MMP14 (5′-caggcaaagcugaugcagauu-3′, Qiagen), PAR-1 (5′-aaggcuacuaugccuacuacuuu-3′, Thermo Fisher Scientific, Waltham, MA, USA), caveolin-1 (siGENOME SMARTpool, Thermo Scientific), caveolin-2 (siGENOME SMARTpool, Thermo Scientific) or non-targeting siRNA (siGENOME SMARTpool, Thermo Scientific) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions.

Techniques: Immunoprecipitation, Confocal Laser Scanning Microscopy, Western Blot, Expressing, Two Tailed Test

Journal: Molecular Cancer

Article Title: Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

doi: 10.1186/s12943-014-0279-8

Figure Lengend Snippet: Exogenous heparan sulfate influences subcellular localisation of caveolin-2. (A) Detergent-resistant membrane (DRM) preparations were made from MDA-MB231 lysates, some treated with 20 μg/ml heparan sulfate for 24 h. Nine fractions were collected from each centrifuge tube and western blotted for flotillin as a marker of DRM pools, transferrin receptor as a marker of detergent soluble membrane proteins (non-DRM), and caveolin-2. The caveolin-2 is almost exclusively in the DRM pool of control cells, while it is displaced to the non-DRM pool when cells are treated with heparan sulfate. Syndecan-2 is reduced after heparan sulfate treatment (B) Caveolin-2 staining of untreated (Ctrl) MDA-MB231 cells and cells treated with 20 μg/ml heparan sulfate (HS) for 24 h before fixation. While the HS-treated cells are more spread than control cells, there is no obvious alteration in the localisation of caveolin-2. Bar = 25 μm.

Article Snippet: Cells were transfected with siRNA targeting syndecan-1 (sc-36587, Santa Cruz Biotechnology, CA, USA), syndecan-2 (sc-41045, Santa Cruz Biotechnology, CA, USA), syndecan-4 (5′-ggccgauacuucuccggaguu-3′, Qiagen, Frederick, MD, USA), MMP14 (5′-caggcaaagcugaugcagauu-3′, Qiagen), PAR-1 (5′-aaggcuacuaugccuacuacuuu-3′, Thermo Fisher Scientific, Waltham, MA, USA), caveolin-1 (siGENOME SMARTpool, Thermo Scientific), caveolin-2 (siGENOME SMARTpool, Thermo Scientific) or non-targeting siRNA (siGENOME SMARTpool, Thermo Scientific) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions.

Techniques: Western Blot, Marker, Staining

Journal: Molecular Cancer

Article Title: Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

doi: 10.1186/s12943-014-0279-8

Figure Lengend Snippet: Syndecan-2 expression correlates with metastatic status in patient. (A) Normal breast tissues (Normal) and breast cancer patient tissues (Cancer) from tissue microarrays were stained for syndecan-2 or caveolin-2 on serial sections. Magnified images are shown in lower panels, bars = 100 μm. (B) Intensity per area for syndecan-2 or caveolin-2 staining was quantified for each core in tissue microarray to compare normal breast tissue and patient breast tissue of different tumour grades, n = 161 for syndecan-2 and n = 165 for caveolin-2. Bar graph was plotted in ln scale. (C) Breast tissue from patients diagnosed with Grade II carcinoma and metastatic lymph node tissue from the same patient were stained for syndecan-2 and caveolin-2. Higher magnification images are shown in lower panels, bars = 100 μm. (D) Quantification data of intensity per area of syndecan-2 or caveolin-2 staining from (C) , n = 26 for syndecan-2 cases and n = 28 for caveolin-2 cases. Bar graph was plotted in ln scale. Error bars = s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, n.s.: not significant, tested by one-way ANOVA with Bonferroni post-hoc test. (E) Levels of syndecan-2 and caveolin-2 staining from serial sections of the 47 triple negative breast carcinoma cases were significantly correlated. r: Spearman’s correlation coefficient = 0.328, p = 0.024.

Article Snippet: Cells were transfected with siRNA targeting syndecan-1 (sc-36587, Santa Cruz Biotechnology, CA, USA), syndecan-2 (sc-41045, Santa Cruz Biotechnology, CA, USA), syndecan-4 (5′-ggccgauacuucuccggaguu-3′, Qiagen, Frederick, MD, USA), MMP14 (5′-caggcaaagcugaugcagauu-3′, Qiagen), PAR-1 (5′-aaggcuacuaugccuacuacuuu-3′, Thermo Fisher Scientific, Waltham, MA, USA), caveolin-1 (siGENOME SMARTpool, Thermo Scientific), caveolin-2 (siGENOME SMARTpool, Thermo Scientific) or non-targeting siRNA (siGENOME SMARTpool, Thermo Scientific) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Microarray