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: Nature communications

Article Title: Multiplatform molecular profiling uncovers two subgroups of malignant peripheral nerve sheath tumors with distinct therapeutic vulnerabilities.

doi: 10.1038/s41467-023-38432-6

Figure Lengend Snippet: Fig. 5 | SHH pathway is important for malignant transformation in a subset of MPNSTs and a therapeutic target. a Capillary-based immunoassay (WES) con- firming knockout of PTCH1 and subsequent increased GLI1 activity in HSC1-λ cells. b Trypan blue counts of parental HSC1-λ and PTCH1-knockout clone. Error bars, s.e.m.; n = 3 biologically independent experiments. c PTCH1-knockout in HSC1-λ cell lines leads to upregulation of SHH pathway (GLI1, GLI2 and SMO). Error bars, s.e.m.; n = 3 biologically independent experiments. d Protein blot confirming knockout of PTCH1 in ipNF06.2A cells. e Trypan blue counts of a parental ipNF06.2A cells and PTCH1-knockout clone. Error bars, s.e.m; n = 3 biologically independent experiments. f PTCH1-knockout in ipNF06.2A leads to upregulation of SHH pathway (GLI1, GLI2 and SMO). Error bars, s.e.m; n = 3 biologically indepen- dent experiments. g Representative images and quantitative cell migration in HSC1-

Article Snippet: Primary antibodies for B-Actin(1:1000, Cat #8457S, Cell Signaling Technologies), Vinculin(1:30000, Cat #V9264, Sigma Aldrich), PTCH1(1:500,Cat#MAB41051, R&D systems) and APC(1:500, Cat #15270, Abcam) were used.

Techniques: Transformation Assay, Knock-Out, Activity Assay, Migration

Journal: Molecular Cancer

Article Title: Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish

doi: 10.1186/1476-4598-8-40

Figure Lengend Snippet: Elevated phospho-AKT1 and Patched 1 levels in zebrafish tumors . A, B, a 3-week-old transgenic fish with trunk tumor (B) and its age-matched tumor-free fish (A). C, D, a 12-week-old fish with an eye tumor (D) and its age-matched tumor free fish (C). Immunofluorescence was done on cryosections for the above fish. E-H, an astrocytoma from a double transgenic fish showed elevated levels of both Patched 1 (F) and phosphorylated AKT (H). Immunofluoresence was done on paraffin sections for this tumor. Negative controls for Patched 1 (E) and phosphorylated AKT (G) were treated the same way except no primary antibodies were added. N, Notochord; AKT-P, phosphorylated AKT. Scale bars, 100 μm for A-D, 50 μm for E-H.

Article Snippet: A rabbit anti-GFP polyclonal antibody (Invitrogen, 1:500), a rabbit anti-phospho-AKT (Thr308) antibody (C31E5, Cell Signalling Technology, 1:200), and a goat anti-Patched 1 (zebrafish) antibody (Everest Biotech, 1:200) were used for immunofluorescence studies of the tumors.

Techniques: Transgenic Assay, Immunofluorescence