Journal: Poultry Science

Article Title: Effect of Angelica Sinensis extract on the angiogenesis of preovulatory follicles (F1–F3) in late-phase laying hens

doi: 10.1016/j.psj.2022.102415

Figure Lengend Snippet: Effect of AS extract on the expression of CD31 in the theca layers. (A–C) Immunofluorescence (IF) showed the expression of CD31 in preovulatory follicles (F1–F3). Scale bar: 100 μm. (D–F) The relative fluorescence intensity of CD31 in preovulatory follicles (F1–F3) was quantified. (G–L) Western blot detected the expression of CD31 in preovulatory follicles (F1–F3). All experiments were performed in triplicate, and the data are the mean ± S.E.M (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Following blocking with skimmed milk powder, membranes were incubated with primary antibodies to protein to detect CD31 (1:1000, ABclonal, Wuhan, China; A0378), VEGFA (1:200, ABclonal, Wuhan, China; A12303), VEGFR (1:500, ABclonal, Wuhan, China; A11127), P-VEGFR (1:500, ABclonal, Wuhan, China; AP0382), HIF1α (1:1000, ABclonal, Wuhan, China; A0378), β-actin (1:2000, ABclonal, Wuhan, China; AC038).

Techniques: Expressing, Immunofluorescence, Fluorescence, Western Blot

Journal: Experimental Hematology & Oncology

Article Title: Fibronectin promotes tumor angiogenesis and progression of non-small-cell lung cancer by elevating WISP3 expression via FAK/MAPK/ HIF-1α axis and activating wnt signaling pathway

doi: 10.1186/s40164-023-00419-w

Figure Lengend Snippet: Fibronectin positively regulated the angiogenesis in NSCLC. ( A ) In vitro Matrigel assay was performed to detect the vascular structure formation in H460 and H1299 cells treated with or without dish-coated fibronectin (10 µg/mL) for 24 h. ( B ) H460 and H1299 cells were treated with or without dish-coated fibronectin (10 µg/mL) for 24 h. Then, Western blot was performed for the detection the proteins of VEGF, CD31, Tie2 and Ve-cadherin. β-actin was used as a loading control. Quantitative analysises of proteins expression were shown in the histogram. ( C ) IHC assay was used to determine the expression of fibronectin and CD31 in tumor tissues from xenograftsderived from H1299 cells mixed with or without fibronectin, and representative data were shown. Data were presented by mean ± SD from three independent experiments. *P < 0.05; **P < 0.01 vs. control

Article Snippet: After electrophoresed on 8–15% SDS-PAGE gels, proteins were transferred to PVDF membranes (Millipore), and blocked with 5% skim milk for 2 h. Then, the membrane was incubated with the primary antibodies against fibronectin (1:1000, Proteintech, 15613-1-AP), WISP3(1:800, Proteintech, 21259-1-AP), p-FAK (1:1000, abcam,ab81298), FAK (1:1000, Proteintech, 66258-1-Ig), p-MEK (1:1000, CST, 9154 S), MEK (1:1000, proteintech, 11049-1-AP), p-ERK (1:1000, CST, 4370 S), ERK (1:1000–1:2000,ABclonal, A16686), p-β-catenin (1:1000, proteintech, 28772-1-AP), β-catenin (1:1000, proteintech, 51067-2 -AP), N-cadherin (1:500, proteintech, 22018-1-AP), vimentin (1:1000, proteintech, 10366-1-AP), OCT4 ( 1:1000-1:5000, proteintech, 11263-1-AP), Nanog (1:1000, CST, 4903s), VEGF (1:1000, ABclonal, A12303), CD31 (1:500, Proteintech, A0378), SOX2 (1:1000, CST, #14,962), Tie2 (1:500, Proteintech, 19157-1-AP), Ve-cadherin (1:1000, Proteintech, 66804-1-Ig), and HIF-1a (1:2000, Proteintech, 20960-1-AP)at 4°C overnight, β-actin was used as a loading control.

Techniques: In Vitro, Matrigel Assay, Western Blot, Control, Expressing

Journal: Experimental Hematology & Oncology

Article Title: Fibronectin promotes tumor angiogenesis and progression of non-small-cell lung cancer by elevating WISP3 expression via FAK/MAPK/ HIF-1α axis and activating wnt signaling pathway

doi: 10.1186/s40164-023-00419-w

Figure Lengend Snippet: WISP3 promoted tube forming potential of NSCLC cells in vitro. ( A ) In vitro Matrigel assay was performed to detect the vascular structure formation in H460 and H1299 cells transfected WISP3 overexpression plasmid (pCDH-WISP3) or empty vector (pCDH) for 24 h. ( B ) H460 and H1299 cells were transfected with pCDH or pCDH-WISP3 for 24 h. Then, Western blot was performed for the detection the proteins of p-β-catenin and β-catenin. β-actin was used as a loading control. Quantitative analysis of p-β-catenin and β-catenin expression were shown in the histogram. ( C ) H460 and H1299 cells were transfected with pCDH or pCDH-WISP3 for 24 h. Then, Western blot was performed for the detection the proteins of VEGF, CD31, Tie2 and Ve-cadherin. β-actin was used as a loading control. Quantitative analysis of proteins expression were shown in the histogram. Data were presented by mean ± SD from three independent experiments. *P < 0.05; **P < 0.01 vs. control

Article Snippet: After electrophoresed on 8–15% SDS-PAGE gels, proteins were transferred to PVDF membranes (Millipore), and blocked with 5% skim milk for 2 h. Then, the membrane was incubated with the primary antibodies against fibronectin (1:1000, Proteintech, 15613-1-AP), WISP3(1:800, Proteintech, 21259-1-AP), p-FAK (1:1000, abcam,ab81298), FAK (1:1000, Proteintech, 66258-1-Ig), p-MEK (1:1000, CST, 9154 S), MEK (1:1000, proteintech, 11049-1-AP), p-ERK (1:1000, CST, 4370 S), ERK (1:1000–1:2000,ABclonal, A16686), p-β-catenin (1:1000, proteintech, 28772-1-AP), β-catenin (1:1000, proteintech, 51067-2 -AP), N-cadherin (1:500, proteintech, 22018-1-AP), vimentin (1:1000, proteintech, 10366-1-AP), OCT4 ( 1:1000-1:5000, proteintech, 11263-1-AP), Nanog (1:1000, CST, 4903s), VEGF (1:1000, ABclonal, A12303), CD31 (1:500, Proteintech, A0378), SOX2 (1:1000, CST, #14,962), Tie2 (1:500, Proteintech, 19157-1-AP), Ve-cadherin (1:1000, Proteintech, 66804-1-Ig), and HIF-1a (1:2000, Proteintech, 20960-1-AP)at 4°C overnight, β-actin was used as a loading control.

Techniques: In Vitro, Matrigel Assay, Transfection, Over Expression, Plasmid Preparation, Western Blot, Control, Expressing

Journal: Experimental Hematology & Oncology

Article Title: Fibronectin promotes tumor angiogenesis and progression of non-small-cell lung cancer by elevating WISP3 expression via FAK/MAPK/ HIF-1α axis and activating wnt signaling pathway

doi: 10.1186/s40164-023-00419-w

Figure Lengend Snippet: Knockdown of WISP3 blocked Fibronectin-promoted cell migration, invasion, angiogenesis and cancer stemness in NSCLC cells. ( A ) H1299 cells were transfected with siNC or siWISP3 for 24 h. Then, the migration and invasion ability of the transfected cells treated with or without dish-coated fibronectin were detected by transwell assay. Quantitative analysis of migrated and invaded cells was shown in the histogram. ( B ) In vitro Matrigel assay was performed to detect the vascular structure formation in H1299 cells treated as ( A ). ( C ) Western blot assay was used to detect the proteins of N-catenin, vimentin, OCT4, SOX2 and Nanog in H1299 cells as treated in ( A ). β-actin was used as a loading control. Quantitative analysis of proteins expression were shown in the histogram. ( D ) Western blot assay was used to detect the proteins of VEGF, CD31, Tie2 and Ve-cadherin in H1299 cells treated as ( A ). β-actin was used as a loading control. Quantitative analysis of proteins expression were shown in the histogram. Data were presented by mean ± SD from three independent experiments. *P < 0.05; **P < 0.01 vs. control

Article Snippet: After electrophoresed on 8–15% SDS-PAGE gels, proteins were transferred to PVDF membranes (Millipore), and blocked with 5% skim milk for 2 h. Then, the membrane was incubated with the primary antibodies against fibronectin (1:1000, Proteintech, 15613-1-AP), WISP3(1:800, Proteintech, 21259-1-AP), p-FAK (1:1000, abcam,ab81298), FAK (1:1000, Proteintech, 66258-1-Ig), p-MEK (1:1000, CST, 9154 S), MEK (1:1000, proteintech, 11049-1-AP), p-ERK (1:1000, CST, 4370 S), ERK (1:1000–1:2000,ABclonal, A16686), p-β-catenin (1:1000, proteintech, 28772-1-AP), β-catenin (1:1000, proteintech, 51067-2 -AP), N-cadherin (1:500, proteintech, 22018-1-AP), vimentin (1:1000, proteintech, 10366-1-AP), OCT4 ( 1:1000-1:5000, proteintech, 11263-1-AP), Nanog (1:1000, CST, 4903s), VEGF (1:1000, ABclonal, A12303), CD31 (1:500, Proteintech, A0378), SOX2 (1:1000, CST, #14,962), Tie2 (1:500, Proteintech, 19157-1-AP), Ve-cadherin (1:1000, Proteintech, 66804-1-Ig), and HIF-1a (1:2000, Proteintech, 20960-1-AP)at 4°C overnight, β-actin was used as a loading control.

Techniques: Knockdown, Migration, Transfection, Transwell Assay, In Vitro, Matrigel Assay, Western Blot, Control, Expressing

Journal: Journal of Gastrointestinal Oncology

Article Title: The long noncoding RNA RMRP-miR-3135a-SV2A axis promotes the development of hepatocellular carcinoma

doi: 10.21037/jgo-2025-259

Figure Lengend Snippet: LncRNA RMRP could regulate SV2A expression through sponging miR-3135a. (A) The expression of miR-3135a was detected via qRT-PCR in HCCLM3 cells with lncRNA RMRP knockdown or overexpression. (B) The expression of miR-3135a was detected via qRT-PCR in HepG2 cells with lncRNA RMRP knockdown or overexpression. (C) A dual-luciferase reporter assay was performed to confirm the interaction between lncRNA RMRP and miR-3135a. (D) A dual-luciferase reporter assay was performed to confirm the interaction between SV2A and miR-3135a. (E,F) The mRNA levels of SV2A were detected in HCCLM3 and HepG2 cells with or without treatment of miR-3135a mimic. (G) The protein expression of SV2A was detected in five HCC tissue samples and five normal tissue samples. (H) The protein expression of SV2A was detected in HCCLM3 and HepG2 cells with lncRNA RMRP knockdown or overexpression. (I) The protein expression of SV2A was detected in HCCLM3 and HepG2 cells with lncRNA RMRP knockdown, miR-3135a inhibition, or a combination of both. Data are presented as the mean and standard deviation. *, P<0.05; **, P<0.01; ***, P<0.001. HCC, hepatocellular carcinoma; Inhibitor, HCC cells transfected with inhibitor of miR-3135a for miR-3135a knockdown; lncRNA, long noncoding RNA; lnc-RMRP-MUT, promotor of lncRNA RMRP with indicated mutation; lnc-RMRP-WT, wild type promotor of lncRNA RMRP; Mimic, HCC cells transfected with Mimic of miR-3135a for miR-3135a overexpression; NC, HCC cells transfected with empty lentivirus; NC-miR, HCC cells transfected with negative control vector; qRT-PCR, quantitative real-time polymerase chain reaction; RMRP, HCC cells transfected with lncRNA RMRP overexpression lentivirus; shCtrl, HCC cells transfected with negative control lentivirus; shRMRP, HCC cells transfected with lncRNA RMRP knockdown lentivirus; SV2A-MUT, promotor of SV2A with indicated mutation; SV2A-WT, wild type promotor of SV2A.

Article Snippet: Antibodies against P53 (1:2,000; cat. no. 10442-1-AP; Proteintech), caspase-3 (1:1,000; cat. no. 19677-1-AP; Proteintech), cleaved caspase-3 (1:1,000; cat. no. BM4620; Boster Bio), Bcl-XL (1:1,000; cat. no. 10783-1-AP; Proteintech), SV2A (1:1,000; cat. no. A03752-3; Boster Bio), and GAPDH (1:30,000; cat. no. 60004-1-Ig; Proteintech) were used as the primary antibodies for overnight incubation at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Over Expression, Luciferase, Reporter Assay, Inhibition, Standard Deviation, Transfection, Mutagenesis, Negative Control, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: The long noncoding RNA RMRP-miR-3135a-SV2A axis promotes the development of hepatocellular carcinoma

doi: 10.21037/jgo-2025-259

Figure Lengend Snippet: LncRNA RMRP could regulate HCC development through the miR-3135a-SV2A axis. (A,B) The overexpression efficiency of SV2A was evaluated through qRT-PCR and WB assays. (C) The CCK-8 assay was performed on HCC cells with the indicated treatment to reveal the regulation of HCC cell proliferation via the lncRNA RMRP/SV2A axis. (D) The CCK-8 assay was performed on HCC cells with the indicated treatment to assess the regulation of HCC cell proliferation via the miR-3135a-SV2A axis. (E) The expression correlation between lncRNA RMRP and immune checkpoints, including CD274 (PD-L1), PDCD1LG2 (PD-L2), and CTLA4, was analyzed based on the TCGA database. Data are presented as the mean and standard deviation. **, P<0.01; ***, P<0.001. HCC, hepatocellular carcinoma; lncRNA, long noncoding RNA; miR-3135a mimic, HCC cells transfected with Mimic of miR-3135a for miR-3135a overexpression; NC, HCC cells transfected with empty lentivirus; OD, optical density; shCtrl, HCC cells transfected with negative control lentivirus; shRMRP, HCC cells transfected with lncRNA RMRP knockdown lentivirus; SV2A (SV2A-OE), HCC cells transfected with SV2A overexpression lentivirus.

Article Snippet: Antibodies against P53 (1:2,000; cat. no. 10442-1-AP; Proteintech), caspase-3 (1:1,000; cat. no. 19677-1-AP; Proteintech), cleaved caspase-3 (1:1,000; cat. no. BM4620; Boster Bio), Bcl-XL (1:1,000; cat. no. 10783-1-AP; Proteintech), SV2A (1:1,000; cat. no. A03752-3; Boster Bio), and GAPDH (1:30,000; cat. no. 60004-1-Ig; Proteintech) were used as the primary antibodies for overnight incubation at 4 °C.

Techniques: Over Expression, Quantitative RT-PCR, CCK-8 Assay, Expressing, Standard Deviation, Transfection, Negative Control, Knockdown