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: Biology Open

Article Title: Formation of the sacrum requires down-regulation of sonic hedgehog signaling in the sacral intervertebral discs

doi: 10.1242/bio.035592

Figure Lengend Snippet: Expression of SHH targets and notochordal markers in mouse sacral NP cells with aging. (A-H) Immunofluorescence and quantification of notochordal/ NP markers: SHH (A,B); CK19 (C,D); BRA (E,F); PTCH1 (G,H) in the S1/S2, S2/S3 (quantification data only) and S3/S4 discs of male FVB mice at P4, 4 weeks, 12 weeks, 14 weeks, and 1 year of age. N =3 each. Scale bars: 100 μm. Nuclei are counter-stained with DAPI in all images. Mean±s.em. Two-way ANOVA. † P <0.05, †† P <0.01, ††† P <0.001 compares data under bars, * P <0.05, ** P <0.01, *** P <0.001 compares data under asterisk to its P4 value.

Article Snippet: Briefly, following permeabilization using either buffered 0.5% Triton ×-100, or 10 mM Sodium Citrate for 20 min at room temperature, sections were blocked in blocking buffer for 1 hour and incubated with a specific primary antibody [GFP, 1:200, 11122, Life Technologies; CK19, 1:100, TROMA-II, Developmental Studies Hybridoma Bank (Iowa City, USA); SHH, 1:25, S4944, Sigma-Aldrich; PTCH1, 1:20, MAB41051, R&D Systems (Minneapolis, USA); Collagen1, 1:100, GTX41285, Genetex (Irvine, USA); CHSO4, 1:100, ab11570, Abcam; Ki67, 1:75, ab156611, Abcam; Brachyury, 1:10, ab156611, Abcam; CD31 (PECAM-1), 1:50, AF3628, R&D Systems] overnight at 4°C in a humidified chamber.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Biology Open

Article Title: Formation of the sacrum requires down-regulation of sonic hedgehog signaling in the sacral intervertebral discs

doi: 10.1242/bio.035592

Figure Lengend Snippet: Constitutive activation of SmoM2 in the NP cells re-activates the sacral disc. (A) Coronal section of S1/S2 discs from CK19 CreERT2/+ ; R26 TOM/+ gavaged at 11 weeks of age and analyzed 1 week later. Red cells are the CK19 CreERT2/+ expressing cells that have undergone recombination (white arrows). (B) H and E staining of the mid-coronal section from the control (CTRL- R26 SmoM2-YFP/SmoM2-YFP ) and SmoM2 ( CK19 CreERT2/+; R26 SmoM2-YFP/SmoM2-YFP ) littermates. (C-F) Quantification of morphometric parameters disc height (C); area occupied by the NP cells (D); the number of NP cells (E); and the number of layers in the AF (F) in the sacral discs of the SmoM2 group compared to littermate controls. (G,H) Immunostaining and quantification of YFP+ NP cells in SmoM2 and control discs. (I-P) Data from immunostaining for SHH and its downstream targets and their quantification: SHH (I,J); PTCH1 (K,L); CK19 (M,N); CHSO4 (O); COL1 expression in S1/S2 discs from controls and SmoM2 mice (P). (Q,R) Immunostaining and quantification of PECAM-1 positive structures in the AF and EP of control and SmoM2 mouse discs. Mean±s.d. N =4 Controls; N =3 SmoM2. Unpaired two-tailed t -test. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. Scale bars: 100 μm. Nuclei are counter-stained with DAPI in A, G, I, K, M, O, P and Q. A is captured using DIC filter.

Article Snippet: Briefly, following permeabilization using either buffered 0.5% Triton ×-100, or 10 mM Sodium Citrate for 20 min at room temperature, sections were blocked in blocking buffer for 1 hour and incubated with a specific primary antibody [GFP, 1:200, 11122, Life Technologies; CK19, 1:100, TROMA-II, Developmental Studies Hybridoma Bank (Iowa City, USA); SHH, 1:25, S4944, Sigma-Aldrich; PTCH1, 1:20, MAB41051, R&D Systems (Minneapolis, USA); Collagen1, 1:100, GTX41285, Genetex (Irvine, USA); CHSO4, 1:100, ab11570, Abcam; Ki67, 1:75, ab156611, Abcam; Brachyury, 1:10, ab156611, Abcam; CD31 (PECAM-1), 1:50, AF3628, R&D Systems] overnight at 4°C in a humidified chamber.

Techniques: Activation Assay, Expressing, Staining, Immunostaining, Two Tailed Test