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: Microorganisms

Article Title: Hungatella hathewayi : A Tumor-Derived Bacterium Enriched in Colorectal Cancer Tissues and a Potential Diagnostic Biomarker

doi: 10.3390/microorganisms14030707

Figure Lengend Snippet: H. hathewayi -derived succinate upregulates HIF-1α and SUCNR1 expression and promotes metastasis by inducing EMT in HCT15 cells. ( A ) Relative mRNA expression of SUCNR1 in HCT15 cells treated with SA or HHM by qPCR. HCT15 cells were cultured in cell medium with 5% HHM, GAM, and SA (positive control) for 24 h. ( B ) Relative mRNA expression of HIF-1α in HCT15 cells treated with 1 mM SA or HHM by qPCR. Cells were cultured under conditions described in ( A ). Untreated cells served as the blank control. ( C – E ) Relative mRNA expression of CDH1 , Vimentin and Snail1 in HCT15 cells treated with SA or HHM by qPCR. Cells were cultured under conditions described in ( A ). Untreated cells served as the blank control. ( F ) Analysis of CDH1, HIF-1α, Snail1, and Vimentin protein expression in HCT15 cells by Western blot. HCT15 cells were cultured in cell medium with 5% HHM, GAM, and SA (positive control) for 24 h. HHM, the culture medium supernatant of H. hathewayi . GAM, bacterial culture medium. SA, 1 mM succinate. Data were from independent experiments and shown as mean ± SD. Compared using Student’s t -test between two groups, and differences among the groups were calculated using One Way ANOVA, followed by Dunnett’s multiple comparison test. Differences among the groups were calculated using One Way ANOVA. * p < 0.05, ** p < 0.01 and **** p < 0.0001.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies targeting NLRP3 (Cat No. 30109-1-AP, ProteinTech Group, Inc., Rosemont, IL, USA), IL-1β (516288, ZEN-BIOSCIENCE Co., Ltd., Chengdu, China), ASC (Cat No. 10500-1-AP, ProteinTech Group, Inc.), Caspase-1 (Cat No. 22915-1-AP, ProteinTech Group, Inc.), IL-18 (Cat No. 10663-1-AP, ProteinTech Group, Inc.), HIF-1α (Cat. PB9253, Boster Biological Technology Co., Ltd., Wuhan, China), CDH1 (Cat. PTM-6222, PTM BioLab Inc., Hangzhou, China), Vimentin (Cat. PTM-5376, PTM BioLab Inc.), Snail1 (Cat. 3879, Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin (Cat. PTM-5455, PTM BioLab Inc.), followed by a 1 h incubation with secondary antibodies (Cat No. SA00001-2, ProteinTech Group, Inc.) at room temperature.

Techniques: Derivative Assay, Expressing, Cell Culture, Positive Control, Control, Western Blot, Comparison