Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: Sequences of primers.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Sequencing, Amplification

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA and FGF2 are upregulated in pancreatic cancer tissues and cell lines. (A) Comparison of HPA and FGF2 expression in pancreatic cancer tissues and adjacent normal pancreatic tissues by immunohistochemical stainin. (B) Analysis of relative mRNA expression levels of HPA and FGF2 in pancreatic cancer and normal pancreatic cell lines by reverse transcription-quantitative PCR. *P<0.05, **P<0.01 vs. HPDE6c7. HPA, heparanase; FGF2, fibroblast growth factor 2; NC, negative control.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Comparison, Expressing, Immunohistochemical staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: Expression of heparanase and fibroblast growth factor 2 in pancreatic cancer tissues and adjacent normal tissues.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: Association between heparanase and fibroblast growth factor 2 expression, and clinicopathological characteristics of patients with pancreatic cancer.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA upregulates the expression of FGF2 in pancreatic cancer cell lines. (A) Analysis of HPA silencing and overexpression efficiency by RT-qPCR. (B) Analysis of HPA silencing and overexpression efficiency by western blot assay. (C) Analysis of FGF2 expression after silencing or overexpressing HPA by RT-qPCR. (D) Analysis of FGF2 expression after silencing or overexpressing HPA by western blotting. β-actin was used as an internal control. *P<0.05, **P<0.01, ***P<0.001. RT-qPCR, reverse transcription-quantitative PCR; HPA, heparanase; FGF2, fibroblast growth factor 2; sh, short hairpin (RNA; specific for HPA); Vector, PANC-1 cell line transfected with empty plasmid containing the GFP sequence.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Transfection, Sequencing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA regulates FGF2 expression via the HPA/SDC1 axis in pancreatic cancer cell lines. (A) RT-qPCR comparing the relative expression levels of SDC1 mRNA in pancreatic cancer lines and the normal pancreatic cell line HPDE6c7. *P<0.05, **P<0.01 vs. HPDE6c7. (B) Detection of the effect of HPA on SDC1 mRNA expression by RT-qPCR. (C) Expression of FGF2 was determined by RT-qPCR analysis after adding the SDC1 inhibitor synstatin to the HPA overexpression group. **P<0.01. RT-qPCR, reverse transcription-quantitative PCR; HPA, heparanase; FGF2, fibroblast growth factor 2; sh, short hairpin (RNA); SDC1, syndecan-1; Vector, PANC-1 cell line transfected with empty plasmid containing the GFP sequence.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Transfection, Sequencing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA promotes cell migration and invasion by activating the PI3K/Akt signaling pathway and EMT processes via FGF2 upregulation. (A) Expression of Palladin protein in various pancreatic cancer cell lines was analyzed by western blot assay. β-actin was used as an internal control. (B) Analysis of the relative expression of Palladin mRNA in pancreatic cancer cell lines by reverse transcription-quantitative PCR. (C) Western blot analysis of the effect of FGF2 on Palladin and Akt. (D) Protein expression of Palladin and Akt. β-actin was used as an internal control. (E) Effect of FGF2, AZD4547 and LY294002 on the migratory ability of PANC-1 cells assessed by wound healing assay. Magnification, ×200. (F) Transwell assays were performed to determine the invasive ability of PANC-1 cells treated with FGF2, AZD4547 and LY294002. (G) Number of invaded PANC-1 cells treated with FGF2, AZD4547 and LY294002. CON, PANC-1 cell line transfected with empty plasmid. (H) Western blot analysis of the effect of HPA and FGF2 on EMT. Vector, PANC-1 cell line transfected with empty plasmid. β-actin was used as an internal control. *P<0.05, **P<0.01, ***P<0.001. HPA, heparanase; FGF2, fibroblast growth factor 2; EMT, epithelial-mesenchymal transition; CON, PANC-1 cell line transfected with empty plasmid; Vector, PANC-1 cell line transfected with empty plasmid containing the GFP sequence.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Migration, Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Wound Healing Assay, Transfection, Plasmid Preparation, Sequencing

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 1. Tagging of the CYP4F2 and CYP4F3 loci. Position and linkage disequilibrium (LD) of genotyped (tag-)SNPs across the CYP4F2 (left) and the CYP4F3 locus (right). The position of (tag-)SNPs on the genes is indicated at the top of the figure. Tall black boxes are coding exons. Short black boxes are untranslated exons. LD is represented by shades of grey as a function of r2 values (black diamonds for r2 ≥0.90, white diamonds for r2 = 0).

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques:

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 3. Impact of rs2108622 and rs3093105 on CYP4F2 protein and mRNA levels. CYP4F2 protein and mRNA levels were measured in ABCD1-KD cell lines (n = 4 per variant) seeded simultaneously and grown under similar conditions. CYP4F2 protein levels were markedly lower upon expression of p.433M variants (A and B). The p.W12G SNP did not affect CYP4F2 protein levels (B). The CYP4F2 mRNA levels were not affected by rs2108622 or rs3093105 (C). Data are presented as mean ± SD. ***p b 0.001 by one-way ANOVA followed by Bonferroni's multiple comparison test. NS is not significant.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Variant Assay, Expressing, Comparison

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 4. CYP4F2 p.V433M decreases the clearance of VLCFA by reducing their ω-oxidation. VLCFA homeostasis was assessed in ABCD1-KD (n = 6) (A) and PEX13-KD (n = 6) (B and C) cell lines exposed to D3-C22:0. For each cell line 3 biological replicates were included. ω-OH-C22:0 and ω-OH-C24:0 are markers for the ω-oxidation of D3-C22:0 and D3-C24:0, respectively; while D3-C24:0, D3-C26:0 reflect the elongation of D3-C22:0. (A) Assessment of the effect of CYP4F2 expression in the ABCD1-KD cell lines showed a non-significant decrease in the amount of the elongation products D3-C24:0 and D3-C26:0 from D3-C22:0. (B) Expression of CYP4F2 p.433 V resulted in a significant decrease in D3-C22:0 elongation to D3-C24:0 and D3-C26:0 in PEX13-KD cells, and (C) significant higher levels of ω-OH-C22:0 and ω-OH-C24:0. Fatty acid levels are in nmol/mg protein. Data are presented as mean ± SD. ***p b 0.001 by unpaired student's t-test.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Expressing

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 5. p.V433M decreases the ω-oxidation of C26:0. The ω-oxidation of C26:0 was measured in ABCD1-KD (n = 6) (A) and PEX13-KD (n = 6) (B and C) and cell lines exposed to D4-C26:0. For each cell line 3 biological replicates were included. (A) Increased ω-oxidation of D4-C26:0 was observed in all ABCD1-KD cell lines expressing CYP4F2 but this effect was more pronounced upon expression of p.433V variants. (B and C) Increased ω-oxidation of C26:0 was observed in all PEX13-KD cell lines expressing CYP4F2, but this effect was more pronounced upon expression of p.433V variants. PEX13-KD cell lines that expressed CYP4F2 p.433V produced significantly higher levels of both ω-OH-C26:0 (B) and C26:0- dicarboxylic acid (C26:0-DCA) (C) than PEX13-KD cell lines expressing CYP4F2 p.433M. n.d. = not detected. Fatty acid levels are in nmol/mg protein. Data are presented as mean ± SD. **p b 0.01 and ***p b 0.001 by unpaired student's t-test.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Expressing, Produced

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 6. p.V433M does not affect the catalytic activity of CYP4F2 in vitro. Microsomes isolated from clones expressing either CYP4F2 p.433V (n = 4) (●) or p.433M (n = 4) (■) were used to determine the kinetic parameters, Km and Vmax, of CYP4F2 for both C22:0 and ω-OH-C22:0. Omega oxidation activity for an increasing concentration of C22:0 (A) or ω-OH-C22:0 (B) was determined and used to calculate the kinetic parameters with the Michaelis-Menten equation. The insert shows the Lineweaver-Burk plot for the collected data. Omega oxidation activities (V) are in nmol/min/mg protein. Substrate concentrations (S) are in μM. Values represent the mean and error bars represent the SD. (C) Differences in CYP4F2 content of microsomes were expressed as a ratio between two variants, using calregulin as a loading control. ***p b 0.001 by unpaired student's t-test.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Activity Assay, In Vitro, Isolation, Clone Assay, Expressing, Concentration Assay, Control

Journal: Biochimica et biophysica acta

Article Title: CYP4F2 affects phenotypic outcome in adrenoleukodystrophy by modulating the clearance of very long-chain fatty acids.

doi: 10.1016/j.bbadis.2016.07.006

Figure Lengend Snippet: Fig. 7. Increased proteasomal degradation of CYP4F2 p.433M. (A) The effect of CYP4F2 p.433V and p.433M on protein stability was assessed by exposure of microsomes isolated from cells (n = 4) expressing CYP4F2 p.433V or p.433M at 50 °C for different time periods followed by CYP4F2 enzyme activity measurement using 200 μM C22:0 as substrate. (B) ABCD1-KD Flp-In 293 cells expressing CYP4F2 p.433V or p.433M were cultured for 24 h in the presence of bortezomib at different concentrations (0, 10, 30 or 100 nM) and CYP4F2 protein levels were determined by Western blotting.

Article Snippet: Crane, GriffithUniversity, Brisbane, Australia, 1:1000), rabbit anti-human CYP4F2 (Fitzgerald Industries International, 1:8000), mouse anti-β-actin (A5441, Sigma, 1:20,000 dilution) or goat anti-human calregulin (sc-6467, Santa Cruz, 1:2500), followed by incubationswith the appropriate IRDye secondary antibody (LI-COR Inc., 1:10,000).

Techniques: Isolation, Expressing, Activity Assay, Cell Culture, Western Blot