Journal: Nature

Article Title: Structures of human Patched and its complex with native palmitoylated Sonic Hedgehog

doi: 10.1038/s41586-018-0308-7

Figure Lengend Snippet: a, Primary structure of Ptch1. Residues 619–720 and 1189–1447 were removed in Ptch1*. b, Hh signaling in Ptch1−/− MEFs transfected with Ptch1 or Ptch1* and response to wild-type or mutant Shh-N ligand via luciferase activity. Shh-N in conditioned medium and transiently expressed Ptch1 were detected by western blotting. c, Pull-down assay of his-tagged Shh-N or native Shh-N with Ptch1* at different molar ratios detected by Coomassie-staining. The assay was reproduced three times with the similar results. d, Palmitoylated Shh-N stimulates Hh signaling but unmodified Shh-N does not. Shh-Light II cells were treated with various concentrations of Shh-N varients, and Hh signaling was measured using luciferase activity. Data (panels b and d) are mean ± s.d. (n=3).

Article Snippet: The expression of Ptch1 variants and internal calnexin in MEFs cells were detected by Western blotting with anti-Ptch1 antibody (GeneTex, 83771) and anti-calnexin antibody (Novus, NB100–1965).

Techniques: Transfection, Mutagenesis, Luciferase, Activity Assay, Western Blot, Pull Down Assay, Staining

Journal: Nature

Article Title: Structures of human Patched and its complex with native palmitoylated Sonic Hedgehog

doi: 10.1038/s41586-018-0308-7

Figure Lengend Snippet: a, Ribbon representation of the structure horizontal to the membrane. Flexible linkers are indicated by dots. b, Structural comparison of transmembrane domains of Ptch1* and NPC1 (pdb code: 5U74) viewed from extracellular side. c, Structural comparison of transmembrane domains of Ptch1* and AcrB trimer (pdb code: 1IWG). One subunit of AcrB is yellow, while the rest are gray. d, SSD comparison of Ptch1* and NPC1 (pdb code: 5U74) in a similar view as the right panel of a. Red arrows indicate shifted helices. Surface representation of the unidentified molecule in yellow. e, Overall structure of ECD-I and ECD-II. f, Interface between ECD-I and ECD-II. Hydrophilic interactions are indicated by dots and residues colored as in panel e.

Article Snippet: The expression of Ptch1 variants and internal calnexin in MEFs cells were detected by Western blotting with anti-Ptch1 antibody (GeneTex, 83771) and anti-calnexin antibody (Novus, NB100–1965).

Techniques: Membrane, Comparison

Journal: Nature

Article Title: Structures of human Patched and its complex with native palmitoylated Sonic Hedgehog

doi: 10.1038/s41586-018-0308-7

Figure Lengend Snippet: a, Ribbon representation of the structure horizontal to the membrane with Ptch1* colored as in Fig. 2 and Shh-N colored in cyan. An Shh-N bound zinc atom is indicated by a gray sphere. Putative endogenous molecules from the cryo-EM density map are shown at 5σ level in red mesh. b, Structural comparison of the membrane domains of apo-Ptch1* (gray) and Ptch1*–Shh-N (colored). c, The palmitate-binding site. d, Interface between Np of Shh-N and ECD-I compared with apo-Ptch1*. e, Secondary interface between Shh-N and ECD-I subdomain 2 compared with apo-Ptch1*. Red arrows represent structural shifts.

Article Snippet: The expression of Ptch1 variants and internal calnexin in MEFs cells were detected by Western blotting with anti-Ptch1 antibody (GeneTex, 83771) and anti-calnexin antibody (Novus, NB100–1965).

Techniques: Membrane, Cryo-EM Sample Prep, Comparison, Binding Assay

Journal: Nature

Article Title: Structures of human Patched and its complex with native palmitoylated Sonic Hedgehog

doi: 10.1038/s41586-018-0308-7

Figure Lengend Snippet: a, The palmitoylated N-terminus of Shh-N is important for Ptch1* binding. A 2:1 Ptch1*:Shh-N molar ratio was used for assays. b, Calcium facilitates the binding between Ptch1 and N-terminal tagged Shh-N. c, Complex of Shh-N and 5E1 (pdb code: 3MXW), with interaction areas on Shh-N in red. Calcium in the interface are indicated by green balls. d, 5E1 blocks the binding of Ptch1* to His-tagged Shh-N but not to native Shh-N. e, Mutagenesis of the secondary interface of Shh-N. f, Mutagenesis of the interface of Ptch1*. The assays (panels a, b, d, e and f) were reproduced three times with the similar results. g, Repression of Hh signaling by Ptch1-AAAA and its response to Shh-N ligand. Shh-N in conditioned medium was shown in Fig. 1d. Hh activity was measured by luciferase assay and data are mean ± s.d. (n=3). The protein was detected by Coomassie-staining (panel a, b and d) or by western blotting (panels e, f and g).

Article Snippet: The expression of Ptch1 variants and internal calnexin in MEFs cells were detected by Western blotting with anti-Ptch1 antibody (GeneTex, 83771) and anti-calnexin antibody (Novus, NB100–1965).

Techniques: Binding Assay, Mutagenesis, Activity Assay, Luciferase, Staining, Western Blot

Journal: Nature

Article Title: Structures of human Patched and its complex with native palmitoylated Sonic Hedgehog

doi: 10.1038/s41586-018-0308-7

Figure Lengend Snippet: a, Complex of Shh-N and Cdo-Fn3 (pdb code: 3D1M) with interaction areas on Shh-N in red. b, Complex of Shh-N and Ihog-Fn3 (pdb code: 2IBG) with interaction areas on Shh-N in red. Hypothetical model of Ptch1*–Shh-N–Cdo complex or Ptch1*–Shh-N-Ihog complex was generated by docking Shh-N-Cdo-Fn3 or Shh-N-Ihog-Fn3 to the Ptch1*– Shh-N structure. c, Model of putative collaboration between Shh-N–Ptch1 and co-receptors or Ptch itself in Hh signaling.

Article Snippet: The expression of Ptch1 variants and internal calnexin in MEFs cells were detected by Western blotting with anti-Ptch1 antibody (GeneTex, 83771) and anti-calnexin antibody (Novus, NB100–1965).

Techniques: Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Hedgehog signaling promotes sorafenib resistance in hepatocellular carcinoma patient-derived organoids

doi: 10.1186/s13046-020-1523-2

Figure Lengend Snippet: GANT61 reversed the increase in Hedgehog signaling proteins and CD44 caused by sorafenib treatment. a Representative bright-field images of immunohistochemical staining for CD44, Gli1, Ptch1 and Shh in patient tumor samples showing low or high expression. Scale bar: 50 μm. b The result of immunohistochemical staining in a box diagram. c Immunofluorescence images of HCC PDOs after treatment with DMSO or GANT61 for 6 days. The cells were stained for CD44 (green) and stained with DAPI (blue). d The levels of CD44 and Hedgehog-related proteins in cell lines (SNU423 and BEL7402) grown as monolayer cells or spheroids. e Western blot analysis showed the levels of CD44 and Hedgehog-related proteins in cell lines (SNU423 and 7402) after treatment with DMSO (control), sorafenib, GANT61 or a combination of sorafenib and GANT61. f Western blot analysis showed the expression of MEKK2 and MEKK3 after sorafenib treatment at different concentrations in cell lines (SNU423 and BEL7402). g Illustration of the GANT inhibits Gli1to reverse the resistance of sorafenib in HCC. (*p < 0.05, ** p < 0.01, *** p < 0.001, P < 0.05 is considered statistically significant)

Article Snippet: The following primary antibodies were used: anti-AFP (Proteintech, 14,550–1-AP), anti-GPC3 (Abcam, ab129381), anti-EpCAM (Proteintech, 21,050–1-AP), anti-Ki-67 (CST, 9449), anti-CD44 (CST, 3570), anti-Sonic Hedgehog (Abcam, ab53281), anti-PTCH1 (Immunoway, YT3598), anti-Smoothened (Abcam, ab113438), and anti-GLI1 (Abcam, ab49314).

Techniques: Immunohistochemical staining, Staining, Expressing, Immunofluorescence, Western Blot