Journal: Clinical Cancer Research

Article Title: Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma

doi: 10.1158/1078-0432.ccr-17-2923

Figure Lengend Snippet: Figure 1. Cholesterol biosynthesis is enhanced in Hh subtype of medulloblastoma. A, A schematic diagram of cholesterol biosynthesis pathway with genes encoding corresponding enzymes listed along arrows. Enzymes: HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1; HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase; MVD mevalonate diphosphate decarboxylase; FDPS farnesyl diphosphate synthase; FDFT1 farnesyl-diphosphate farnesyltransferase 1; SQLE squalene epoxidase; LSS lanosterol synthase; NSDHL NAD(P) dependent steroid dehydrogenase-like; DHCR7 7-dehydrocholesterol reductase; DHCR24 24-dehydrocholesterol reductase. Statins inhibit conversion of HMG-CoA to mevalonate through competitive inhibition of HMG-CoA reductase (HMGCR). Triparanol blocks the last step of cholesterol biosynthetic pathway through inhibition of 24-dehydrocholesterol reductase (DHCR24). B and C, Boxplots showing the enrichment scores from GSVA. x and y-axes illustrate enrichment statistics across 4 different subtypes of medulloblastoma. Positive and negative scores indicate positive and negative enrichment, respectively. Pink dots represent the enrichment score for each sample in that subtype. Black bar in the middle of the box indicates median. D, Heatmaps showing expression of representative genes in the Hh and cholesterol synthesis pathways. z-scores calculated for each gene are plotted on a red (higher expression) and blue (low expression) scale. Top color bar, subtype. E–G, IHC analysis of NSDHL (E), ABCA1 (F), and SREBP2 (G) proteins in mouse Ptch1þ/ medulloblastoma. Normal adjacent tissue represents a cerebellar lobe with differentiated granule neurons used as a control. Scale bar, 100 mm; inset, 10 mm.

Article Snippet: Membranes were probed with antibodies against Gli1 (Cell Signaling Technology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1- AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000), GAPDH (Sigma-Aldrich, G8795, 1:1,000).

Techniques: Inhibition, Expressing, Control

Journal: Clinical Cancer Research

Article Title: Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma

doi: 10.1158/1078-0432.ccr-17-2923

Figure Lengend Snippet: Figure 2. Deficiency of NSDHL inhibits Ptch1þ/ medulloblastoma cell proliferation. A, Western blot analysis of NSDHL and Gli1 protein expression in Ptch1þ/ medulloblastoma cells following infection with lentivirus carrying Nsdhl shRNA (#3 and #4) or scrambled shRNA and uninfected control after 48 hours in culture. GAPDH was used as a loading control. B and C, Immunofluorescence analysis of BrdUrd among Ptch1þ/ medulloblastoma cells infected with scrambled control shRNA (mCherryþ, B) or Nsdhl shRNA #4 (mCherryþ, C) after 48 hours in culture. D, Percentage of BrdUrdþ cells in infected medulloblastoma cells (mCherryþ) in Fig. 2B and C. E and F, Sagittal sections of medulloblastoma tissue from Math1-CreERT2/R26R-eYFP/Ptch1þ/ mice were immunostained against Zic1 and YFP (E), S100b, and YFP (F). G, Western blot analysis of NSDHL and Gli1 protein expression in medulloblastoma tissue from Math1-CreERT2/Nsdhlfl/fl/Ptch1þ/ and Nsdhlfl/fl/Ptch1þ/

Article Snippet: Membranes were probed with antibodies against Gli1 (Cell Signaling Technology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1- AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000), GAPDH (Sigma-Aldrich, G8795, 1:1,000).

Techniques: Western Blot, Expressing, Infection, shRNA, Control

Journal: Clinical Cancer Research

Article Title: Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma

doi: 10.1158/1078-0432.ccr-17-2923

Figure Lengend Snippet: Figure 3. Cholesterol is required for Smo activation in Hh signal transduction. A, Western blot analysis of Gli1 protein expression in Ptch1þ/ medulloblastoma (MB) cells following treatment with simvastatin alone or in combination with WSC for 48 hours. B, Western blot analysis of Gli1 protein expression in NIH 3T3 cells following treatment with Hh-CM, simvastatin, triparanol, or together with WSC for 48 hours. C and D, Western blot analysis of Gli1 protein expression in Ptch1fl/flMEFs (C) or SmoM2 MEFs (D) infected with a lentivirus carrying Cre recombinase or control empty vector following treatment with simvastatin, triparanol, or WSC for 48 hours. E, Western blot analysisof Gli1and Ptch1 proteins in NIH 3T3 cells infected with a lentivirus carrying a Gli1-HA construct or an empty vector following treatment with simvastatin, triparanol, or WSC for 48 hours. GAPDH was used as a loading control. F, A schematic diagram of Hh signaling pathway. In the absence of Hh ligand, its receptor Ptch1 tethers Smo, preventing the downstream activation of Hh pathway. After interaction with Hh, Ptch1 releases Smo, causing activation of Gli1/2 transcription factor and leading to transcription of Hh pathway genes (Gli1, Ptch1, etc.). Cholesterol is required for Smo activation in Hh signal transduction.

Article Snippet: Membranes were probed with antibodies against Gli1 (Cell Signaling Technology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1- AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000), GAPDH (Sigma-Aldrich, G8795, 1:1,000).

Techniques: Activation Assay, Transduction, Western Blot, Expressing, Infection, Control, Plasmid Preparation, Construct

Journal: Clinical Cancer Research

Article Title: Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma

doi: 10.1158/1078-0432.ccr-17-2923

Figure Lengend Snippet: Figure 4. Cholesterol biosynthesis inhibitors suppress proliferation of Ptch1þ/ medulloblastoma cells. A–K, Medulloblastoma cells isolated from Ptch1þ/ mouse were cultured in the presence of vehicle (A) or simvastatin(B–D), triparanol (E), or together with WSC (F–J) for 48 hours, before cultured cellswere immunostained against Ki67 (red) and counterstained with DAPI. Scale bar, 10 mm. K, Percentage of Ki67-positive cells in cultured Ptch1þ/ medulloblastoma cells in the presence of simvastatin, triparanol, or with addition of WSC. Significance asterisk key: P < 0.05 (), P < 0.01 (), P < 0.001 (), P < 0.0001 ().

Article Snippet: Membranes were probed with antibodies against Gli1 (Cell Signaling Technology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1- AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000), GAPDH (Sigma-Aldrich, G8795, 1:1,000).

Techniques: Isolation, Cell Culture

Journal: Clinical Cancer Research

Article Title: Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma

doi: 10.1158/1078-0432.ccr-17-2923

Figure Lengend Snippet: Figure 5. Simvastatin inhibits growth of Ptch1þ/

Article Snippet: Membranes were probed with antibodies against Gli1 (Cell Signaling Technology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1- AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000), GAPDH (Sigma-Aldrich, G8795, 1:1,000).

Techniques:

Journal: Clinical Cancer Research

Article Title: Statins Synergize with Hedgehog Pathway Inhibitors for Treatment of Medulloblastoma

doi: 10.1158/1078-0432.ccr-17-2923

Figure Lengend Snippet: Figure 6. Statins synergize with vismodegib in inhibiting Ptch1þ/ medulloblastoma (MB) growth. A and B, Growth rate (A) and size (B) of subcutaneous Ptch1þ/

Article Snippet: Membranes were probed with antibodies against Gli1 (Cell Signaling Technology, CST2534, 1:1,000), NSDHL (Proteintech, 15111-1- AP, 1:1,000), Ptch1 (Novus Biologicals, MAB41051, 1:1,000), GAPDH (Sigma-Aldrich, G8795, 1:1,000).

Techniques:

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: Immunohistochemical staining of PTCH1 protein in (A) HGSC, (B) LGSC, (C) healthy ovarian tissue and (D) healthy fallopian tube tissue. Red arrows point to the epithelium, while blue arrows point to the stroma (scale bar, 100 µ m; magnified windows, magnification, ×400). (E) Total, (G) cytoplasmic and (I) nuclear expression of PTCH1 protein in the tumor epithelium of HGSCs and LGSCs and epithelium of healthy ovaries and fallopian tubes, and (F) total, (H) cytoplasmic and (J) nuclear expression of PTCH1 protein in the connective stroma of HGSCs and LGSCs and ovarian and fallopian tube stroma, determined by immunohistochemical analysis (H-score). H score (n) + H score (c) is a sum of H-scores calculated for the nuclear and cytoplasmic protein expression. * P<0.05, ** P<0.0001. HGSC, high-grade serous ovarian carcinoma; LGSC, low-grade serous ovarian carcinoma; PTCH1, protein patched homolog 1; n, nuclear; c, cytoplasmic.

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

Techniques: Immunohistochemical staining, Staining, 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: Immunofluorescence staining of PTCH1 protein in high-grade serous ovarian carcinoma cell lines, OVCAR8 and OVSAHO, and normal fallopian tube non-ciliated epithelial cell line FNE1. (A, first row) Staining with an anti-PTCH1a antibody. (A, second row) Nuclei stained with Hoechst. (A, third row) Merged images (scale bars, 10 µ m). (B) Mean fluorescence intensity of PTCH1 protein in OVCAR8, OVSAHO and FNE1 cell lines. ** P<0.0001. PTCH1, protein patched homolog 1.

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

Techniques: Immunofluorescence, Staining, Fluorescence

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: Immunofluorescence staining of PTCH1 protein in high-grade serous ovarian carcinoma cell lines, OVCAR8 and OVSAHO, and normal fallopian tube non-ciliated epithelial cell line FNE1. (A, first row) Staining with an anti-PTCH1d antibody. (A, second row) Nuclei stained with Hoechst. (A, third row) Merged images (scale bars, 10 µ m). (B) Mean fluorescence intensity of PTCH1 protein in OVCAR8, OVSAHO and FNE1 cell lines. * P<0.05, ** P<0.0001. PTCH1, protein patched homolog 1.

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

Techniques: Immunofluorescence, Staining, Fluorescence

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: Subcellular localization of PTCH1 protein in high-grade serous ovarian carcinoma cell lines, OVCAR8 and OVSAHO, and normal fallopian tube non-ciliated epithelial cell line FNE1. (A) Efficiency of protein extraction from different cellular compartments was verified by examining the expression of GAPDH protein in the cytoplasmic protein fraction, Na + /K + ATPase in the membrane fraction, fibrillarin in the nuclear soluble protein fraction, H3K4me2 protein in the chromatin-bound protein fraction and β-actin in the cytoskeletal protein fraction. Western blotting using (B) anti-PTCH1a and (C) anti-PTCH1d antibodies. PTCH1, protein patched homolog 1; input, total proteins; cytopl., cytoplasmic proteins; membr., membrane proteins; nucl., nuclear soluble proteins; chrom., chromatin-bound proteins; cytosk., cytoskeletal proteins; NTF; N-terminal cytoplasmic fragment; aa, amino acids.

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

Techniques: Protein Extraction, Expressing, Membrane, Western Blot

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: Methylation-specific PCR analysis of the PTCH1 gene in serous ovarian carcinomas and ovarian cancer cell lines. (A) Representative images of methylation-specific PCR analysis for PTCH1 gene promoter in healthy OV and FT tissues, LGSC and HGSC. (B) DNA promoter methylation status of the PTCH1 gene in HGSC cell lines, OVCAR8 and OVSAHO, and normal fallopian tube non-ciliated epithelial cell line FNE1. M, methylated reaction; UM, unmethylated reaction; MC, methylated human control; UMC, unmethylated human control; OV, ovarian; FT, fallopian tube; HGSC, high-grade serous ovarian carcinoma; LGSC, low-grade serous ovarian carcinoma.

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

Techniques: Methylation, Control

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