Journal: Journal of Cancer

Article Title: RUNX1 regulates the proliferation and chemoresistance of colorectal cancer through the Hedgehog signaling pathway

doi: 10.7150/jca.51338

Figure Lengend Snippet: RUNX1 decreased 5-FU sensitivity of CRC cells via Hedgehog signaling pathway. A. 5-FU sensitivity measured by CCK8 assays in HCT116/RUNX1, SW480/shRUNX1, HCT116/Vector and SW480/Scramble cells with the different concentrations of 5-FU treated; error bars, SD. B. Apoptosis rate measured by flow cytometer in HCT116/RUNX1, SW480/shRUNX1, HCT116/Vector and SW480/Scramble cells with 5-FU treated; error bars, SD. C. Expression of RUNX1 in HCT116, SW480 and RKO cell lines with 5-FU treated. D. Expression of ABCG2 in HCT116/RUNX1, SW480/shRUNX1, HCT116/Vector and SW480/Scramble groups. E. Expression levels of GLI1 PTCH1 ABCG2 mRNA in HCT116/Vector and HCT116/RUNX1, SW480/scramble and SW480/shRUNX1 group; error bars, SD. F. HCT116 and RKO cell proliferation determined by CCK8 assay in Vector and RUNX1, vector with GDC and RUNX1 with GDC groups G. Expression of ABCG2 and targeted genes activated by Hedgehog signaling pathway in HCT116/Vector, HCT116/RUNX1, HCT116/RUNX1 with GDC groups.

Article Snippet: Protein lysates were prepared, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride (PVDF) membranes, and blotted according to standard methods using antibodies to the following: RUNX1 (#4336, CST), PTCH1 (#2468, CST), PTCH2 (#2470, CST), GLI1 (#3538, CST), Shh (#2207, CST), ABCG2 (#42078, CST) and GAPDH (60004-1-Ig, Proteintech).

Techniques: Plasmid Preparation, Flow Cytometry, Expressing, CCK-8 Assay

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-PTCH1c , PTCH1 , Rabbit polyclonal , 1-80 , IHC, 1:300; IF, 1:100; WB, 1:1,000 , OASG05688; Aviva Systems Biology, Corp..

Techniques: Immunohistochemistry-IF

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 1. Analysis of Hh pathway activation in human MPM specimens and rat MPM model in tumor and stromal fractions. A, immunohistochemical detection of GLI1, DHH, PTCH1, and HHIP in tumor (T) and stroma (S) and box plot of semiquantitative expression levels (H-score) of GLI1 and DHH in 146 MPM specimens, and PTCH1 and HHIP in 8 MPM specimens (lines indicate medians). B, histology of rat MPM model revealed cellular areas of closely packed spindled tumor cells (T) with relatively large, irregular nuclei, adjacent to stroma (S) containing fibroblasts (, organized small spindle cells), blood vessels (X), and inflammatory cells (small round cells with dense blue chromatin; arrow). Of note is the presence of some fibroblasts and inflammatory cells also in the tumor area. Scatter plot shows semiquantitative expression levels of GLI1, DHH, PTCH1, and HHIP (H-score) of 6 tumors (lines indicate medians).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Activation Assay, Immunohistochemical staining, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 2. The comparison of Hh pathway activation in vitro and in vivo rat MPM model. A, expression levels (mRNA) of pathway components were monitored in IL45-luc cultured in 10% serum, primary cell culture in 3% O2 without serum compared with tumor from the rat model (in vivo). B, cell growth measurement by MTT (left) and colony formation assay (right) shows that IL45-luc and the parental line (IL45) cultured in 20% O2 with serum are identically sensitive to vismodegib (data are given in mean SD; , P < 0.05; , P < 0.001; , P < 0.0001). C and D, IL45 parental cells were exposed to increasing concentration of vismodegib and measured for the downregulation of hedgehog pathway target gene (Gli1 and Ptch1) in addition to Gli2 and Gli3. No significant suppression of all genes was observed upon the treatment course (24 and 48 hours).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Comparison, Activation Assay, In Vitro, In Vivo, Expressing, Cell Culture, Colony Assay, Concentration Assay

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 4. Efficient downregulation of Hh signaling and reduction of tumor cell growth following the treatment with vismodegib. A, mRNA levels of Hh target genes, Gli1 is strongly reduce in skin and tumor of vismodegib-treated rats. Expression level of other GLI1 target genes, Ptch1 and Hhip, isalso reduced in treated tumor compared with control. Dhh, Gli2, and Gli3 expression remains unchanged. B, immunohistochemical staining of GLI1 and HHIP showing reduced expression (intensity and frequency) in treated compared with control. More GLI1- and HHIP-negative stromal cells are present in the treated rat (see circles: T, tumor; S, stroma). C, quantitative analysis histo(H)-score of GLI1, HHIP, PTCH1, and DHH staining showing pronounced GLI1 and HHIP downregulation in stromal fractions. D, proliferation (Ki-67–positive), mitotic (phospho-histone H3–positive) indices are significantly reduced in the treated group. No change in necrosis and apoptosis level (TUNEL-positive nuclei) is observed between groups. (C, control, n ¼ 6; T, treated, n ¼ 6, data are given in mean SD; , P < 0.05; , P < 0.001).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Expressing, Control, Immunohistochemical staining, Staining, TUNEL Assay

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 5. Vismodegib dose dependently suppresses growth of mesothelioma cells but could not suppress Hh pathway in vitro. A, primary cells isolated from four different rat tumors cultured in 3% O2 without serum and IL45-luc cell line cultured 20% O2 were exposed to increasing concentration of vismodegib and measured for viability. No difference in terms of sensitivity to vismodegib was observed (data are given in mean SD). B, two lines of primary cell (isolated from control rats) were treated with vismodegib for 24 hours and analyzed for the downregulation of Gli1. No significant suppression of Gli1 was observed. C, IC50 of vismodegib determined in four cell lines after 72-hour treatment (MTT assay). D, the expression levels of Hh pathway components (Dhh, Smo, Ptch1, Gli1) of the four cell lines showing no correlation with their sensitivity to vismodegib.

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: In Vitro, Isolation, Cell Culture, Concentration Assay, Control, MTT Assay, Expressing

Journal: Molecular Cancer Therapeutics

Article Title: Antagonizing the Hedgehog Pathway with Vismodegib Impairs Malignant Pleural Mesothelioma Growth In Vivo by Affecting Stroma

doi: 10.1158/1535-7163.mct-15-0583

Figure Lengend Snippet: Figure 6. Daily treatment with vismodegib causes downregulation of previously described fibroblast Hh-responsive genes. A, mRNA levels of canonical Hh target gene such as CyclinD1, Abcg2 remained unchanged in vismodegib-treated group compared with control. Previously described fibroblast Hh-responsive genes, i.e., Fn1, Vegfa, and Lif mRNA levels were reduced in vismodegib-treated group. (C, control, n ¼ 6; T, treated, n ¼ 6; data are given in mean SD; , P < 0.05; , P < 0.001). B, mouse embryonic fibroblast NIH3T3 cells were treated with supernatant collected from primary culture of rat MPM cells. Treatment with SMO agonist (SAG) was employed as a positive control. Gli1, Ptch1, and Fn1 expression levels were analyzed after 72-hour incubation at 37C 3% O2 (data represent mean of triplicates SD).

Article Snippet: Tissues were cut into 3-mm slices and rehydrated through series of graded alcohol followed by IHC using the following antibodies: GLI1 (Santa Cruz Biotechnology, H-300), Fibronectin (Abcam, ab6328), DHH (Santa Cruz Biotechnology, H-19), PTCH1 (Aviva Systems Biology, ARP44249_P050), HHIP (Santa Cruz Biotechnology, H-280), Ki-67 (Cell Marque, CMC27531021), phospho-histone H3 (ser10; Cell Signaling Technology, #9701).

Techniques: Control, Positive Control, Expressing, Incubation

Journal: Open Medicine

Article Title: GPC2 deficiency inhibits cell growth and metastasis in colon adenocarcinoma

doi: 10.1515/med-2022-0421

Figure Lengend Snippet: Prediction of pathways involved in GPC2: (a and b) the genes co-expressed with GPC2 in COAD tissues were enriched in (a) notch signaling and (b) hedgehog signaling and (c) After transfection of shNC, shGPC2 into HCT-8 and SW620 cells, the protein levels of PTCH1, PTCH2, GLI1, NOTCH1, HES1, and DLL4 were detected by Western blotting ( n = 3). KEGG: Kyoto Encyclopedia of Genes and Genomes. ** P < 0.01, *** P < 0.001.

Article Snippet: After separated by using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel, the proteins were transferred onto polyvinylidenefluoride membranes, which were subsequently incubated with 5% skimmed milk at 25°C for 2 h. The membrane was incubated with GPC2 antibody (Biorbyt, catalog number: orb157203), PTCH1 antibody (Biorbyt, catalog number: orb538088), PTCH2 antibody (Biorbyt, catalog number: orb416229), GLI1 antibody (Proteintech, catalog number: 66905-1-Ig), NOTCH1 antibody (Biorbyt, catalog number: orb256723), HES1 antibody (Biorbyt, catalog number: orb36445), DLL4 antibody (Biorbyt, catalog number: orb97478), or glycerine aldehyde phosphate dehydrogenase antibody (Proteintech, catalog number: 10494-1-AP) at 4°C overnight, followed by horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG(H + L) (Proteintech, Catalog number: SA00001-2) or HRP-conjugated goat anti-mouse IgG(H + L) (Proteintech, Catalog number: SA00001-1) at 25°C for 2 h. Finally, Super Sensitive ECL Solution (Beijing Bai’aolaibo Technology Co., Ltd, Catalog number: MT0076) was used to display the results.

Techniques: Transfection, Western Blot