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

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

doi: 10.18632/oncotarget.2778

Figure Lengend Snippet: Figure 1: PTCH1 repression in TMZ-treated GBM cells involved miRNA. U87 and T98G cells were treated with 200 μM TMZ. At 72 h, real time PCR was performed for PTCH1 mRNA (A) and western blot for PTCH1 and SHH (precursor (P) and N-terminus (N)) and β-actin. The values for the untreated group were assigned 1 and are represented by an open bar. The changes in the treated cells were presented as fold change over the vehicle-treated cells. (B) U87 and T98G were knocked down for SHH with siRNA and then analyzed for SHH (precursor, P and mature, N) and β-actin by western blot. (C) The knocked down cells in `C’ were treated with TMZ. At 72 h, the cells were assessed for viability with Cell Titer Blue. The data are presented as the mean % viability relative to vehicle treated siRNA transfected cells ±SD, n = 4. (D) U87 and T98G were knocked down for Dicer1. Control was transfected with non-targeting oligo. Western blot was performed for Dicer. The membrane was stripped and reprobed for β-actin. (E) The cells in `E’ were treated with 200 μM TMZ. At 72 h, western blot was performed for PTCH1. The membrane was stripped and reprobed for β-actin (F) and assessed for viability. (G) p < 0.05 vs. control and non-targeting oligo.

Article Snippet: The reporter gene vector containing the PTCH1 3′ UTR was custom ordered from Origene Technologies (Rockville, MD).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Control, Membrane

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

doi: 10.18632/oncotarget.2778

Figure Lengend Snippet: Figure 2: Increased miR-9 in GBM. (A) Computational analyses were performed for potential miRNA interacting site with the PTCH1 3ʹ UTR. The alignment between the sequence for miR-9 and the 3ʹ UTR of PTCH1 is shown below. (B) U87 and T98G cells were treated with 200 μM TMZ. At various times, real time PCR (Taqman) was performed for miR-9. (C) The studies in `C’ were repeated, except for studies at the 72-h time point, and with primers for miR-9-2, using Sybr-Green. (D) U87 and T98G were transfected with non- targeting oligo or anti-miR-9 and then treated with 200 μM TMZ. After 72 h, cell viability was assessed and presented as % viability, n = 4 ±SD. (E) The heat map shows the analyses from 505 miRNA arrays with GBM tissues from The Cancer Genome Atlas (TCGA). The map represents the miRNAs with +/– 0.5-fold changes from the internal control. Arrow highlights the output for miR-9. *p < 0.05 vs. other time points, **p < 0.05 vs. vehicle. ***p < 0.05 vs. non-targeting oligo.

Article Snippet: The reporter gene vector containing the PTCH1 3′ UTR was custom ordered from Origene Technologies (Rockville, MD).

Techniques: Sequencing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transfection, Control

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

doi: 10.18632/oncotarget.2778

Figure Lengend Snippet: Figure 3: MiR-9 targets PTCH1 and activates SHH signaling and drug transporters. (A & B) CCL64, stably transfected with pPTCH-UTR-JM, was transiently transfected with pre-miR-9, non-targeting oligo and/or target protection (TP) oligo. After 48 h, luciferase activities were quantitated and then presented as normalized luciferase ±SD, n = 4; TP: Target Protector (A). Western blots were performed with whole cell extracts for PTCH1 and β-actin. The normalized band densities are shown at the bottom of the images (B). (C) U87 and T98G, stably transfected with pPTCH-UTR-JM or pPTCH-UTR∆-JM, were transiently transfected with anti-miR-9 and then treated with 200 μM TMZ or vehicle. After 48 h, luciferase activities were quantitated and the results are presented as mean RLU ±SD, n = 4. (D & E) MiR-9 was ectopically expressed in U87 and T98G cells. Total RNA was isolated and then studied by real time PCR for PTCH1 mRNA (D) or Gli1 (E). (F) Diagram show increased miR-9 in TMZ-treated GBM cells leading to activated SHH signaling. (G) Western blots with whole cell extracts were performed for Gli1, PTCH1 and β-actin using miR-9-transfected or vector-transfected GBM cells. (H–K) U87 and T98G cells were ectopically expressed for miR-9 or transfected with vector alone. Real-time PCR was performed for MDR1 (H) and ABCG2 (I). The values for control vector in the real time were normalized to 1 for fold-change of the miR-9 transfectants, mean±SD, n = 4; flow cytometry for membrane MDR1 (J) and ABCG2 (K). *p < 0.05 vs. Pre-mir-9 and TP, **p < 0.05 vs. pPTCH-UTR-JM. ***p < 0.05 vs. vector alone.

Article Snippet: The reporter gene vector containing the PTCH1 3′ UTR was custom ordered from Origene Technologies (Rockville, MD).

Techniques: Stable Transfection, Transfection, Luciferase, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Flow Cytometry, Membrane

Journal: Oncotarget

Article Title: Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level.

doi: 10.18632/oncotarget.2778

Figure Lengend Snippet: Figure 4: Expressions of PTCH1, miR-9-2 and drug transporters in naïve and recurrent GBM cells from patients. (A) Real-time PCR was performed for the miRNA, predicted for PTCH1 (Figure 2A). The results are presented as the mean fold change ±SD, n = 4. (B) Real time PCR for miR-9-2 was performed in four independent studies using RNA from BT145 and BT164 cells. The results of BT145 were normalized to 1 and then used to calculate the fold change for the values of BT164, mean±SD. (C) Real time PCR were performed for PTCH1 as for `B’ and the results are similarly presented. (D) Whole cell extracts from BT145 and BT164 were studied by western blot for PTCH1, SHH-P (precursor), SHH-N (mature) and β-actin. (E) Real time PCR was performed for MDR1 and ABCG2 mRNA with total RNA from BT145 and BT164. (F & G) Flow cytometry was performed for membrane P-glycoprotein (MDR1) (F) and ABCG2 (G). The right panels show the overlay between the two patients. *p < 0.05 vs. BT145.

Article Snippet: The reporter gene vector containing the PTCH1 3′ UTR was custom ordered from Origene Technologies (Rockville, MD).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Membrane