Journal: Molecular Medicine Reports

Article Title: Downregulation of lncRNA ZFAS1 protects H9c2 cardiomyocytes from ischemia/reperfusion-induced apoptosis via the miR-590-3p/NF-κB signaling pathway

doi: 10.3892/mmr.2020.11340

Figure Lengend Snippet: H/R induces cell apoptosis and gene expression changes in H9c2 cells. (A) MTT assay for cell viability following H/R exposure. (B) Apoptotic rate of cells was detected by flow cytometry. (C) Reverse transcription-quantitative PCR was used to detect the expression level of ZFAS1, which was normalized by GAPDH, and of miR-590-5p, which was normalized by U6. (D) Representative western blotting images used to determine protein expression levels of (E) p50, TNF-α and IL-6, and (F) Bax, Bcl-2, pro-caspase-3 and cleaved caspase-3. *P<0.05, **P<0.01, ***P<0.001 vs. 0 h. H/R, hypoxia/reoxygenation; IL, interleukin; miR, microRNA; TNF-α, tumor necrosis factor-α; ZFAS1, ZNFX1 antisense RNA 1.

Article Snippet: Rat H9c2 cardiomyocyte cells (cat. no. CRL-1446; American Type Culture Collection) were cultured with DMEM (cat. no. 12491-15; Thermo Fisher Scientific, Inc.) with 10% of FBS (cat. no. 10100-147; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. 15640055; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO 2 .

Techniques: Gene Expression, MTT Assay, Flow Cytometry, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Molecular Medicine Reports

Article Title: Downregulation of lncRNA ZFAS1 protects H9c2 cardiomyocytes from ischemia/reperfusion-induced apoptosis via the miR-590-3p/NF-κB signaling pathway

doi: 10.3892/mmr.2020.11340

Figure Lengend Snippet: ZAFS1 knockdown reduces H/R-induced H9c2 apoptosis. (A) A WT-ZFAS1 3′UTR luciferase reporter vector and a MUT-ZSAF1 3′UTR luciferase reporter vector with mutations on the miR-590-3p binding sites of the ZFAS1 3′UTR were constructed. (B) RT-qPCR was used to detect the expression levels of ZAFS1 or miR-590-3p in H9c2 cells without H/R induction. (C) miR-590-3p-NC, miR-590-3p-mimic and miR-590-3p-inhibitor were transected into H9c2 cells, and luciferase activity was determined. (D) RT-qPCR results of miR-590-3p expression level in H9c2 cells transfected with si-ZFAS1. (E) Western blotting was used to detect the protein expression levels of Bax, Bcl-2, pro-caspase-3 and cleaved caspase-3 in H9c2 cells after si-ZFAS1 transfection and H/R injury. (F) Flow cytometry results of the percentage of apoptotic cells of different transfected groups following H/R induction. ***P<0.001 vs. si-NC group. 3′UTR, 3′untranslated regions; H/R, hypoxia/reoxygenation; miR, microRNA; MUT, mutation; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; WT, wild-type; ZFAS1, ZNFX1 antisense RNA 1.

Article Snippet: Rat H9c2 cardiomyocyte cells (cat. no. CRL-1446; American Type Culture Collection) were cultured with DMEM (cat. no. 12491-15; Thermo Fisher Scientific, Inc.) with 10% of FBS (cat. no. 10100-147; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. 15640055; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO 2 .

Techniques: Knockdown, Luciferase, Plasmid Preparation, Binding Assay, Construct, Quantitative RT-PCR, Expressing, Activity Assay, Transfection, Western Blot, Flow Cytometry, Mutagenesis, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Downregulation of lncRNA ZFAS1 protects H9c2 cardiomyocytes from ischemia/reperfusion-induced apoptosis via the miR-590-3p/NF-κB signaling pathway

doi: 10.3892/mmr.2020.11340

Figure Lengend Snippet: miR-590-3p targets p50 and reduces H/R-induced apoptosis and inflammation. (A) A WT-p50 3′UTR and a MUT-p50 3′UTR luciferase reporter vector, with mutations on miR-590-3p binding sites of the p50 3′UTR were constructed. (B) miR-590-3p-NC, miR-590-3p-mimic or miR-590-3p-inhibitor were transected into H9c2 cells, and luciferase activity was detected. (C) Protein expression levels of Bax, Bcl-2, pro-caspase-3 and cleaved-caspase-3, and (D) p50, TNF-α and IL-6 were detected by western blotting in H/R-induced H9c2 cells. (E) Flow cytometry results of the percentage of apoptotic cells in different transfected groups with H/R. ***P<0.001 vs. miR-590-3p-NC. 3′UTR, 3′untranslated region; H/R, hypoxia/reoxygenation; IL, interleukin; miR, microRNA; MUT, mutation; NC, negative control; TNF-α, tumor necrosis factor-α; WT, wild-type.

Article Snippet: Rat H9c2 cardiomyocyte cells (cat. no. CRL-1446; American Type Culture Collection) were cultured with DMEM (cat. no. 12491-15; Thermo Fisher Scientific, Inc.) with 10% of FBS (cat. no. 10100-147; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. 15640055; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO 2 .

Techniques: Luciferase, Plasmid Preparation, Binding Assay, Construct, Activity Assay, Expressing, Western Blot, Flow Cytometry, Transfection, Mutagenesis, Negative Control

Journal: Molecular Medicine Reports

Article Title: Downregulation of lncRNA ZFAS1 protects H9c2 cardiomyocytes from ischemia/reperfusion-induced apoptosis via the miR-590-3p/NF-κB signaling pathway

doi: 10.3892/mmr.2020.11340

Figure Lengend Snippet: TNF-α induces H9c2 cell apoptosis in a dose-dependent manner. (A) Apoptotic rate of H9c2 cell was detected by flow cytometry in cells treated with 0, 1, 2 and 4 µg/ml TNF-α for 12 h. (B) MTT was used to detect the viability of H9c2 cells exposed to TNF-α. (C) Western blotting was used to detect the protein expression levels of Bax, Bcl-2, pro-caspase-3 and cleaved-caspase-3. ***P<0.001 vs. 0 µg/ml group, TNF-α, tumor necrosis factor-α.

Article Snippet: Rat H9c2 cardiomyocyte cells (cat. no. CRL-1446; American Type Culture Collection) were cultured with DMEM (cat. no. 12491-15; Thermo Fisher Scientific, Inc.) with 10% of FBS (cat. no. 10100-147; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (cat. no. 15640055; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO 2 .

Techniques: Flow Cytometry, Western Blot, Expressing

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: ChiVMV HCPro directly interacts with CAT1 and CAT3 in vitro and in vivo . (A) Yeast two-hybrid assay. The ability of cells to grow on synthetic dropout medium lacking Leu, Trp, His, and Ade (-LWHA) suggested the interaction. (B) GST pull-down assay showing the interaction among HCPro, CAT1, and CAT3 in vitro . Purified CAT1–MBP, CAT2–MBP, CAT3–MBP, or MBP was incubated with HCPro–GST. After being immunoprecipitated with GST beads, the proteins were detected by protein gel blot analysis with anti-MBP or anti-GST antibodies. (C) BiFC assay. HCPro interacted with CAT1 and CAT3 in N. benthamiana leaves.Scale bars=30 μm.

Article Snippet: To investigate whether CAT1, CAT3, or HCPro could alter ChiVMV infection, overexpressing transgenic lines of CAT1, CAT3 (CAT1-OX and CAT3-OX), and HCPro-OX, and knockout transgenic lines of cat1 , cat3 , and double cat1cat3 ( cat1 -KO, cat3 -KO, and cat1cat3 -KO) were generated ( , ).

Techniques: In Vitro, In Vivo, Y2H Assay, Pull Down Assay, Purification, Incubation, Immunoprecipitation, Western Blot, Bimolecular Fluorescence Complementation Assay

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: Identification of HCPro domains responsible for the interaction between host factors and HCPro. (A) Schematic description of deletion mutants of ChiVMV HCPro. HCPro can be divided schematically into three regions: the N-terminus (residues 1–100), central region (residues 101–300), and C-terminus (residues 301–457). (B) Identification of the interaction specificity between CAT1 and HCPro from different sources. (C) The C-terminus of HCPro is necessary for the interaction. Different purified deletion mutants of HCPro–MBP or MBP were incubated with CAT1–GST or CAT3–GST. After being immunoprecipitated with GST beads, the proteins were detected by protein gel blot analysis with anti-MBP or anti-GST antibodies.

Article Snippet: To investigate whether CAT1, CAT3, or HCPro could alter ChiVMV infection, overexpressing transgenic lines of CAT1, CAT3 (CAT1-OX and CAT3-OX), and HCPro-OX, and knockout transgenic lines of cat1 , cat3 , and double cat1cat3 ( cat1 -KO, cat3 -KO, and cat1cat3 -KO) were generated ( , ).

Techniques: Purification, Incubation, Immunoprecipitation, Western Blot

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: HCPro interacted with CAT1 and CAT3 and inhibited their activities. (A) Gene expression of CAT1 and CAT3 , and (B) their protein accumulations in the HCPro-OX lines and WT plants (using 8-week-old seedlings). (C) CAT1, (D) CAT2, and (E) CAT3 enzyme activity assays in reactions containing varying molar ratios of MBP–CAT to deletion mutants of HCPro. CAT (500 pM) was incubated with or without HCPro deletion mutants at 37 °C for the indicated times. MBP was used as negative control. The full length of HCPro is marked as HCPro. HC-N, amino acids 1–100; HC-C, amino acids 301–457. (F) Catalase activity and (G) H 2 O 2 content in the HCPro-OX lines and WT plants (using 8-week-old seedlings). (H) Necrosis symptoms, (I) coat protein (CP) accumulation, and (J) virus replication of EV, CAT1, and CAT3 introduced into cat1cat3 -KO plants under ChiVMV infection. The circles indicate the area of necrosis. Scale bars=2.5 cm. (K) Catalase activity and (L) H 2 O 2 content in EV, CAT1, and CAT3 introduced into cat1cat3 -KO plants. Fw, fresh weight; EV, empty vector. Values are means and SDs from three biological replicates per genotype and time point. Lower case letters indicate statistically significant differences ( P <0.05).

Article Snippet: To investigate whether CAT1, CAT3, or HCPro could alter ChiVMV infection, overexpressing transgenic lines of CAT1, CAT3 (CAT1-OX and CAT3-OX), and HCPro-OX, and knockout transgenic lines of cat1 , cat3 , and double cat1cat3 ( cat1 -KO, cat3 -KO, and cat1cat3 -KO) were generated ( , ).

Techniques: Gene Expression, Activity Assay, Incubation, Negative Control, Virus, Infection, Plasmid Preparation

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: Overexpression of HCPro and knockout of CAT1 enhanced ChiVMV infection, whereas knockout of CAT3 did not affect ChiVMV infection. (A and C) Symptoms of the mock-inoculated or ChiVMV-infected WT and transgenic plants at 9, 15, and 40 dpi. Scale bars=5 cm (upper panel) and 0.25 cm (lower panel) in (A). Scale bars=10 cm (upper panel), 20 cm (middle panel), 1.5 cm (lower panel) in (C). (B and D) Detection of ChiVMV HCPro and coat protein (CP) in ChiVMV-infected WT and transgenic plants by western blot at 9, 25, and 40 dpi. Systemically infected leaves were collected for detection.

Article Snippet: To investigate whether CAT1, CAT3, or HCPro could alter ChiVMV infection, overexpressing transgenic lines of CAT1, CAT3 (CAT1-OX and CAT3-OX), and HCPro-OX, and knockout transgenic lines of cat1 , cat3 , and double cat1cat3 ( cat1 -KO, cat3 -KO, and cat1cat3 -KO) were generated ( , ).

Techniques: Over Expression, Knock-Out, Infection, Transgenic Assay, Western Blot

Journal: Journal of Experimental Botany

Article Title: Chilli veinal mottle virus HCPro interacts with catalase to facilitate virus infection in Nicotiana tabacum

doi: 10.1093/jxb/eraa304

Figure Lengend Snippet: The local RSS activity of ChiVMV HCPro was not influenced by host factors CAT1 and CAT3. (A and D) Nicotiana benthamiana leaves were infiltrated with a mixture of three A. tumefaciens cultures carrying different constructs, as indicated in the middle panel, and photographed at 3 dpi. (B and E) Western blot analysis of protein extracts from N. benthamiana leaves infiltrated with mixtures of A. tumefaciens carrying different constructs as indicated on the left. The expression of CAT1 and CAT3 was confirmed with CAT antibody. The expression of HCPro and GFP was confirmed with HCPro and GFP antibodies, respectively. Coomassie Brilliant Blue (CBB) staining of the large subunit of Rubisco was used as a loading control. (C and F) qPCR analysis of the GFP mRNA accumulation level. Bars represent the mean and SD of values obtained from three biological repeats. Significant differences ( P <0.05) are denoted by different lower case letters. (G and H) Nicotiana benthamiana leaves were infiltrated with a mixture of three A. tumefaciens cultures and photographed at 5 and 7 dpi.

Article Snippet: To investigate whether CAT1, CAT3, or HCPro could alter ChiVMV infection, overexpressing transgenic lines of CAT1, CAT3 (CAT1-OX and CAT3-OX), and HCPro-OX, and knockout transgenic lines of cat1 , cat3 , and double cat1cat3 ( cat1 -KO, cat3 -KO, and cat1cat3 -KO) were generated ( , ).

Techniques: Activity Assay, Construct, Western Blot, Expressing, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Aqueous Extract of Mori Folium Exerts Bone Protective Effect Through Regulation of Calcium and Redox Homeostasis via PTH/VDR/CaBP and AGEs/RAGE/Nox4/NF-κB Signaling in Diabetic Rats

doi: 10.3389/fphar.2018.01239

Figure Lengend Snippet: MF treatment improved calcium homeostasis through regulation of PTH/VDR/CaBP signaling in the duodenums and kidneys of diabetic rats. (A–D) Show serum levels of P, Ca, 1,25(OH) 2 D 3 and PTH in the different groups of rats. (E–H) Show the representative micro-images of IHC staining (sections were counterstained with hematoxylin; original magnification, × 100, Scale bar: 200 μm; × 200, Scale bar: 100 μm) and their analyses of the levels of VDR in the duodenums and CaBP-28k in the kidneys of different groups of rats. (I–L) Show the representative images of western blot and their analyses of protein expressions of VDR and TRPV6 in the duodenums, and CaBP-28k and TRPV6 in the kidneys of different groups of rats. Data are presented as mean ± SD. n = 10. The dark brown particles denote positive staining. IOD denotes integrated optical density of interested areas. # p < 0.05 or ## p < 0.01 compared with NC group, * p < 0.05 or ** p < 0.01 compared with DM group, ΔΔ p < 0.01 compared with MFL group.

Article Snippet: Anti-TRPV6 polyclonal antibody (Cat. No: 13411-1-AP) was from Proteintech (Rosemont, USA).

Techniques: Immunohistochemistry, Western Blot, Staining

Journal: Frontiers in Pharmacology

Article Title: Aqueous Extract of Mori Folium Exerts Bone Protective Effect Through Regulation of Calcium and Redox Homeostasis via PTH/VDR/CaBP and AGEs/RAGE/Nox4/NF-κB Signaling in Diabetic Rats

doi: 10.3389/fphar.2018.01239

Figure Lengend Snippet: Schematic diagram illustrated the underlying mechanisms of MF aqueous extract in improving bone quality of diabetic rats. Administration of MF aqueous extract to diabetic rats improves calcium homeostasis through increasing the level of 1,25(OH) 2 D 3 and decreasing the level of PTH as well as promoting VDR and TRPV6 expressions in the duodenum and CaBP-28k expression in the kidneys. Meanwhile, MF also rebuilds redox balance through reducing the expressions of AGEs, RAGE, Nox4, and NF-κB in the femurs and tibias of diabetic rats. MF, Mori Folium; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3; PTH, parathormone; VDR, vitamin D receptor; TRPV6, transient receptor potential V6; CaBP-28k, calcium-binding protein-28k; AGEs, advanced glycation end products; RAGE, receptor of advanced glycation end products; Nox4, NADPH oxidoreductase 4; NF-κB, nuclear factor kappa-B.

Article Snippet: Anti-TRPV6 polyclonal antibody (Cat. No: 13411-1-AP) was from Proteintech (Rosemont, USA).

Techniques: Expressing, Binding Assay