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: Developmental biology

Article Title: NFAT5/TonEBP controls early acquisition of notochord phenotypic markers, collagen composition, and sonic hedgehog signaling during mouse intervertebral disc embryogenesis

doi: 10.1016/j.ydbio.2019.07.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Next, the sections were incubated for 1 hour in the appropriate blocking solution (either 5-10% Normal Goat Serum, 10% Fetal Bovine Serum, or reagent from M.O.M.™ Immunodetection Kit; vector Laboratories, BMK-2202) and subsequently incubated overnight at 4°C with primary antibody against Brachyury (1:20; R&D, AF2085), CA3 (1:150; Santa Cruz, sc-50715), vimentin (1:200; Cell Signaling, D21H3), β-actin (1:100; Cell Signaling, 13E5), aggrecan (1:50, MilliporeSigma, AB1031), chondroitin sulfate (1:300; Abcam, ab11570), collagen II (1:400, Fitzgerald, 70R-CR008), collagen I (1:100; Abcam, ab34710), Shh (1: 300, Novus, NBP2-22139), Ptch1 (1:200; R&D, MAB41051), Smo (1:50; Abcam, ab72130), Gli1 (1:250; Abcam, ab151796), and IFT88 (1:250; MilliporeSigma, ABC932).

Techniques: Recombinant, In Situ, Positive Control

Journal: Developmental biology

Article Title: NFAT5/TonEBP controls early acquisition of notochord phenotypic markers, collagen composition, and sonic hedgehog signaling during mouse intervertebral disc embryogenesis

doi: 10.1016/j.ydbio.2019.07.004

Figure Lengend Snippet: NFAT5 null embryos show stage-dependent dysregulation of sonic hedgehog signaling involving nonclassical expression of Gli1. (A) Sonic hedgehog (SHH) in NFAT5 mutants showed elevated expression levels in both the notochord and perinotochordal mesenchyme (PM) at E12.5. (A’, A”) SHH Levels in early NP were similar to wild-type at E13.5 and then markedly reduced at E17.5. (B) Patched-1 (PTCH1) levels in mutants did not mirror elevated SHH in the notochord and PM. (B’, B”) Minimal PTCH1 expression was observed in the mutant NP at E13.5 and E17.5. (C) NFAT5 nulls showed normal levels of smoothened (SMO) in the notochord with decreased levels in the PM. (C’, C”) At E13.5 and E17.5, no differences were seen between groups. (D, D’) In mutants GLI1 levels increased in the notochord and PM at E12.5, and mirrored SHH levels in the early NP at E13.5. (D”) GLI1 levels were refractory to NFAT5 deletion at E17.5. Scale bar = 100 μm. (E-H) Staining of the notochord, PM, and NP was quantified by Area Fraction (% Area). (I) Schematic representing the generation of Ift88 null allele in Shh expressing cells. (J) Timeline showing tamoxifen injection strategy to conditionally knockout Ift88 without disrupting disc development. (K) Agarose gel of PCR-amplified genomic DNA confirming Ift88 floxed alleles. (L) Representative images of sagittal sections of intervertebral discs from E17.5 ShhcreERT2;Ift88f/f (cKO) animals stained by Safranin-O/Fast Green/Hematoxylin compared to controls. (M, M’) Immunostaining and quantification of IFT88-positive NP cells confirmed that IFT88 was knocked out with high efficiency. (N, N’) Gli1 levels were unaffected in ShhcreERT2; Ift88f/f embryos. Sale bars = 20 μm. Quantitative measurements represent mean ± SD. n.s. = not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Next, the sections were incubated for 1 hour in the appropriate blocking solution (either 5-10% Normal Goat Serum, 10% Fetal Bovine Serum, or reagent from M.O.M.™ Immunodetection Kit; vector Laboratories, BMK-2202) and subsequently incubated overnight at 4°C with primary antibody against Brachyury (1:20; R&D, AF2085), CA3 (1:150; Santa Cruz, sc-50715), vimentin (1:200; Cell Signaling, D21H3), β-actin (1:100; Cell Signaling, 13E5), aggrecan (1:50, MilliporeSigma, AB1031), chondroitin sulfate (1:300; Abcam, ab11570), collagen II (1:400, Fitzgerald, 70R-CR008), collagen I (1:100; Abcam, ab34710), Shh (1: 300, Novus, NBP2-22139), Ptch1 (1:200; R&D, MAB41051), Smo (1:50; Abcam, ab72130), Gli1 (1:250; Abcam, ab151796), and IFT88 (1:250; MilliporeSigma, ABC932).

Techniques: Expressing, Mutagenesis, Staining, Injection, Knock-Out, Agarose Gel Electrophoresis, Amplification, Immunostaining

Journal: Developmental biology

Article Title: NFAT5/TonEBP controls early acquisition of notochord phenotypic markers, collagen composition, and sonic hedgehog signaling during mouse intervertebral disc embryogenesis

doi: 10.1016/j.ydbio.2019.07.004

Figure Lengend Snippet: NFAT5-mediated regulation of Shh signaling is cell-autonomous in notochordal-NP cells. (A) mRNA levels of Nfat5, Ptch1, and Smo were significantly suppressed in notochordal-NP cells transduced with Nfat5 shRNA (shNFAT5) compared to control shRNA (shCtr). mRNA levels of Shh were markedly increased in shNFAT5 cells, whereas Gli1 mRNA levels were not affected. (n = 3) (B) Western blot showing three independent experiments showed significantly decreased protein levels of PTCH1 with a downward trend in SMO and no change in GLI1 following Nfat5 knockdown. In contrast to Shh transcript, SHH protein levels remained unaffected in knockdown cells. (C) Predicted TonE binding sites identified by MatInspector (Genomatix Software Suite) in the proximal promoters of Ptch1 and Smo in rat, mouse, and human show high conservation. (E) Activation of canonical SHH signaling in notochordal-NP cells with SMO agonist (SAG) induced Gli1 mRNA but not Nfat5 mRNA levels. (n = 4) (F) Treatment with the SMO inhibitor cyclopamine (CyA) reduced levels of Gli1 mRNA but not Nfat5 mRNA (n = 4). Protein levels of NFAT5 measured by Western blot (G) and corresponding densitometry from three independent experiments (G’) show no effect on expression by both SAG and CyA treatment (n = 3). In G, one representative Western blot is shown. Quantitative measurements represent mean ± SD. n.s. = not significant; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Next, the sections were incubated for 1 hour in the appropriate blocking solution (either 5-10% Normal Goat Serum, 10% Fetal Bovine Serum, or reagent from M.O.M.™ Immunodetection Kit; vector Laboratories, BMK-2202) and subsequently incubated overnight at 4°C with primary antibody against Brachyury (1:20; R&D, AF2085), CA3 (1:150; Santa Cruz, sc-50715), vimentin (1:200; Cell Signaling, D21H3), β-actin (1:100; Cell Signaling, 13E5), aggrecan (1:50, MilliporeSigma, AB1031), chondroitin sulfate (1:300; Abcam, ab11570), collagen II (1:400, Fitzgerald, 70R-CR008), collagen I (1:100; Abcam, ab34710), Shh (1: 300, Novus, NBP2-22139), Ptch1 (1:200; R&D, MAB41051), Smo (1:50; Abcam, ab72130), Gli1 (1:250; Abcam, ab151796), and IFT88 (1:250; MilliporeSigma, ABC932).

Techniques: Transduction, shRNA, Western Blot, Binding Assay, Software, Activation Assay, Expressing

Journal: International Journal of Oncology

Article Title: New insight into the role of PTCH1 protein in serous ovarian carcinomas

doi: 10.3892/ijo.2022.5435

Figure Lengend Snippet: List and specifications of anti-PTCH1 primary antibodies used in IHC, IF and WB.

Article Snippet: Anti-PTCH1d , PTCH1 , Mouse monoclonal , 122-436 , IHC, 1:50; IF, 1:25; WB, 1:500 , NBP1-47945; Novus Biologicals, LLC.

Techniques: Immunohistochemistry-IF

Journal: International Journal of Oncology

Article Title: New insight into the role of PTCH1 protein in serous ovarian carcinomas

doi: 10.3892/ijo.2022.5435

Figure Lengend Snippet: List and specifications of anti-PTCH1 primary antibodies used in IHC, IF and WB.

Article Snippet: Anti-PTCH1d , PTCH1 , Mouse monoclonal , 122-436 , IHC, 1:50; IF, 1:25; WB, 1:500 , NBP1-47945; Novus Biologicals, LLC.

Techniques: Immunohistochemistry-IF