Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: A small population of ACC cells H295R overexpresses Ptch1 at the plasma membrane . ( A ) H295R were labeled with an anti-Ptch1 antibody directed against the extracellular loop and cells presenting Ptch1 at their plasma membrane (H295R-PM-Ptc+ AF594+ cells) were sorted. AF594+ in blue represents the percentage of cells with Ptch1 at the cell surface (H295R-PM-Ptc+ cells). ( B ) Surface labeling of Ptch1 using anti-Ptch1 antibody directed against the extracellular loop of Ptch1 (Alexa 594 in red) on nonpermeabilized parental H295R and H295R-PM-Ptc+ cells. Nuclei were stained with DAPI (in blue). The histogram represents the mean ± SEM of Alexa 594 fluorescence intensity per cell (****: p -value < 0.00005 ( p -value = 2 × 10 −36 )).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Clinical Proteomics, Membrane, Labeling, Staining, Fluorescence

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: H295R-PM-Ptc+ cells are more resistant to chemotherapy than parental cells . ( A ) Doxorubicin (dxr) cytotoxicity. H295R and H295R-PM-Ptc+ cells were treated for 48 h with increasing concentrations of dxr before cell viability measure. ( B ) Doxorubicin IC50 of H295R-PM-Ptc+ and H295R parental cells in the absence or the presence of 10 μM of the Ptch1 efflux inhibitor methiothepin. ( C ) H295R-PM-Ptc+ cells accumulate less doxorubicin than parental H295R cells. Cells on coverslips were incubated with 2 μM dxr for 15, 30, 60, 180 and 240 min and immediately fixed with PFA. Dxr fluorescence was acquired using a filter for Alexa 594 and quantified using ImageJ software. About 100 cells (from three wells) were scored per condition per experiment. All data presented are the mean ± SEM of at least 3 independent experiments. Significance is attained at p -value < 0.05 (*), (**** p < 0.00005).

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Incubation, Fluorescence, Software

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Differentially expressed genes (DEG) between H295R-PM-Ptc+ and parental H295R cells selected for their role in cancer. Genes overexpressed are indicated in red and genes underexpressed are in blue.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Activation Assay, Expressing, Inhibition, Gene Expression, Migration, Marker, Biomarker Discovery

Journal: Pharmaceutics

Article Title: Persistent Properties of a Subpopulation of Cancer Cells Overexpressing the Hedgehog Receptor Patched

doi: 10.3390/pharmaceutics14050988

Figure Lengend Snippet: Composition of active modules containing one or more of the identified genes of interest listed in Table 1 (in bold) with genes upregulated in red and genes downregulated in blue, representative enrichment and role of differentially expressed genes (DEGs) in cancers.

Article Snippet: Cells were collected using Accutase (StemCell), centrifuged and incubated with monoclonal rat anti-Ptch1 antibody (MAB41051 R&D Systems; 10 μg/mL) and then with anti-rat antibody coupled to Alexa 594 in ice in FACS buffer (PBS buffer with FBS 5% and EDTA 2 μM).

Techniques: Migration, Cell Differentiation, Activation Assay, Membrane, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Specification of a dopaminergic phenotype from adult human mesenchymal stem cells.

doi: 10.1634/stemcells.2007-0212

Figure Lengend Snippet: Figure 1. Flow cytometry analysis and bright-field images of MSCs in culture. (A): Human MSCs were isolated from bone marrow (BM) aspirates and analyzed after four cell passages. For (A1–A5), red traces indicate positive signal and green traces indicate negative signal. Isolated MSCs are positive for the MSC markers CD44 (A1) and CD105 (endoglin, SH2) (A2). A subset of MSCs is positive for Stro-1 (A5). MSCs are negative for the primitive hematopoietic marker CD34 (A4) and for the endothelial marker CD31 (A3). MSCs are 71% double positive for both PTCH and SMO (upper right), 2% are single positive for PTCH (upper left), and 16% are single positive for SMO (lower right), whereas 11% are negative for both (lower left) (A6). (B): Bright-field images of human MSCs in culture. Morphological change was observed in human MSCs after induction with SHH, FGF8, and bFGF. Unstimulated, pure population of MSCs isolated from BM aspirates after four cell passages (B1). MSCs induced for 6 days (B2) and 12 days (B3). (C): Control images of MSCs cultured with Neurobasal medium and B27 only. MSCs in culture for 3 days (C1), 6 days (C2), and 12 (C3) days do not show a change in morphology. Abbreviations: bFGF, basic fibroblast growth factor; FGF8, fibroblast growth factor 8; PTCH, patched; SHH, sonic hedgehog; SMO, smoothened.

Article Snippet: Primary antibodies Stro-1 (R&D Systems Inc., Minneapolis, http://www. rndsystems.com), patched (PTCH) (Abcam, Cambridge, U.K., http://www.abcam.com), smoothened (SMO) (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), tyrosine hydroxylase (TH), and III tubulin (Chemicon, Temecula, CA, http://www. chemicon.com) were labeled with secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, http://www. jacksonimmuno.com).

Techniques: Flow Cytometry, Isolation, Marker, Control, Cell Culture

Journal: Biological Research

Article Title: SHh activity and localization is regulated by perlecan

doi: 10.4067/s0716-97602011000100008

Figure Lengend Snippet: Figure 2. Cell associated SHh is altered in perlecan null cells: A) Immunoblot anti-perlecan (left gel) and anti-stub (right gel) of extracts from wt and perlecan-/- cells showed that perlecan null cells have no perlecan protein and other HSPG remain at normal levels.B) Wt and perlecan null cells were seeded and processed for immunofl uorescence with anti-SHh and anti-Ptc1 antibodies post permeabilization (+TX-100) or without permeabilization (-TX-100). Reaction for anti-SHh antibody is lost in perlecan -/- cells after permeabilization, but is not affected in perlecan-/- cells compared to wt cells without detergent. Ptc1 expression is maintained in perlecan-/- fi broblasts. C) SHHAP binding is reduced in perlecan -/- cells. Wt and perlecan -/- cells were grown for 3 days in 96-well plates, washed and incubated with different dilutions of SHHAP fusion protein for 2 hours at 4ºC. Then cells were lysed and bound AP activity and protein concentration determined. Figure shows that perlecan-/- cells bound less SHHAP than wt cells, indicating that perlecan is necessary for SHh binding to cell surface. Wt, wild type; per-/-, perlecan null. Scale Bar= 10 mM.

Article Snippet: Primary antibodies used were: antiperlecan domain II, anti-SHh 5E1 (1/50, Hybridoma Bank), anti- Ptc1 (1/100, Santa Cruz).

Techniques: Western Blot, Expressing, Binding Assay, Incubation, Activity Assay, Protein Concentration