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: Frontiers in Veterinary Science

Article Title: An isocitrate lyase gene-deleted strain of Nocardia seriolae in live attenuated vaccine development against fish nocardiosis

doi: 10.3389/fvets.2025.1664034

Figure Lengend Snippet: Immune response of hybrid snakehead after immunization with the attenuated NS-ΔICL vaccine. (A) Alkaline phosphatase (AKP), (B) acid phosphatase (ACP), (C) peroxidase (POD), (D) lysozyme (LZM) activities, and (E) specific IgM antibody levels in serum at 1, 4, 7, 14, 21, 28, and 35 days post-vaccination (d.p.v.). Data are presented as the mean ± SD ( N = 3 biological replicates; serum samples from three individual fish per group per time point, with each sample measured in technical triplicate). Asterisks indicate significant differences between the NS-ΔICL group and the PBS control group (* p < 0.05).

Article Snippet: Antibody combined with the antigen which was detected by using the rabbit anti-hybrid snakehead IgM antibody previously prepared by our lab. Microplates were incubated with goat anti-rabbit IgG HRP conjugate (BOSTER Biological Technology, China).

Techniques: Control

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Representative image of leptin expression in lung adenocarcinoma. Leptin expression in tumors and paired normal lung tissues was detected by immunochemical staining analysis. (A) Leptin protein was overexpressed in a moderately differentiated lung adenocarcinoma samples with acinar predominant growth compared with normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma (epithelial cells) of lung cancer tissues. (B) There was no difference in the expression of leptin protein in a case of well-differentiated lung adenocarcinoma with lepidic predominant (LPA) growth compared with normal control lung tissue. Leptin was seen to be expressed at low levels in the parenchyma of lung cancer tissue and in normal lung tissue. (C) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma sample with papillary predominant growth (PPA) compared to normal control lung tissue. It can be seen that leptin protein was mainly expressed in the parenchyma of lung cancer tissues. (D) Leptin protein was overexpressed in a poorly differentiated lung adenocarcinoma sample with micropapillary predominant growth (MPA) compared to normal control lung tissue. It can be seen that leptin protein was expressed in both parenchyma and mesenchyme of lung cancer tissues. (E) Leptin protein was overexpressed in a highly differentiated lung adenocarcinoma with solid predominant growth compared to normal control lung tissue. It can be seen that leptin protein was expressed in the parenchyma, mesenchyme, and normal control lung tissues of lung cancer, but the expression level was low in normal tissues. (F) Leptin protein was overexpressed in a lung minimally invasive adenocarcinoma (MIA) compared to normal control lung tissue from the same patient. All experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin-peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope.

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: The effect of endogenous leptin expression on the proliferation of pulmonary adenocarcinoma cells. (A) The clone formation test of H1299 and A549 cells on the effect of proliferation of pulmonary adenocarcinoma; 50 ng/mL artificial recombinant leptin protein was used to simulate the impact of exogenous leptin in circulating blood on tumor cells; (B) the flow cell cycle analysis of PI staining showing the effect of endogenous leptin expression on the cell cycle of lung adenocarcinoma in H1299 and A549 cells; (C) AV/PI double-staining flow cytometry apoptotic cell test showing the effect of endogenous leptin expression in the apoptosis of lung adenocarcinoma in H1299-sh and A549 cells. All the experiments were repeated three times. Immunohistochemistry staining IHC staining of leptin was performed according to the manufacturer’s instructions. A streptavidin–peroxidase staining kit was purchased from ZSGB BIO (Beijing, China). The paraffin-embedded tissues were prepared by a pathology specialist, and dewaxed and rehydrated in the lab. The experimental steps were carried out according to the instructions of the SP kit. The tissues were incubated with primary antibodies diluted to the recommended concentration overnight at 4 °C with antibodies that was dilute to the recommended concentration. Then 3,3'-diaminobenzidine (DAB) staining was performed, and the results were observed under a microscope (100×).

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Recombinant, Cell Cycle Assay, Staining, Double Staining, Flow Cytometry, Immunohistochemistry, Incubation, Concentration Assay, Microscopy

Journal: Annals of Translational Medicine

Article Title: Autocrined leptin promotes proliferation of non-small cell lung cancer (NSCLC) via PI3K/AKT and p53 pathways

doi: 10.21037/atm-20-7482

Figure Lengend Snippet: Molecular mechanisms of endogenous leptin expression effect on the proliferative ability of lung adenocarcinoma cells. (A) Effect of endogenous leptin expression on key signaling molecules of PI3K/AKT pathway and its downstream signaling pathway in H1299 and A549 cells detected by western blotting; (B) effect of endogenous leptin expression on the p53 signaling pathway in A549 cells by western blotting; (C) endogenous leptin expression level on the expression level of autophagy-related protein LC3-II in H1299 cell lines detected by protein immunofluorescence. (D) Effect of endogenous leptin expression on key signaling molecules of the mTOR pathway and its downstream signalings in H1299 and A549 cells detected by western blotting. All the experiments were repeated three times. Scale bar: 50 µm.

Article Snippet: A ntibodys and reagents Rabbit anti-human leptin monoclonal antibody was purchased from Bioss Antibodies (Woburn, MA, USA); rabbit anti-human LC3, β-actin, and P62 monoclonal antibodies were purchased from Proteintech; rabbit anti-human Ob-R monoclonal antibodies were purchased from Shenyang Wanlei Biological Company (Shenyang, China); Rabbit anti-human AKT , p - AKT , p - ERK , p - NF -κ B , p65 , cyclin D1 , CDK2 , p53 , MDM2 , p - MDM2 , and p-mTOR monoclonal antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence