Journal: Animals : an open access journal from MDPI

Article Title: Antiviral Activity of 1-Deoxynojirimycin Extracts of Mulberry Leaves Against Porcine Epidemic Diarrhea Virus.

doi: 10.3390/ani15091207

Figure Lengend Snippet: Figure 2. DNJ inhibits PEDV infection in Vero-E6 cells. (A) The overall design of Vero E6 cells infected and treated with DNJ (25 µM, 50 µM, 100 µM). The green line indicates drug treatment, and the orange line indicates PEDV infection. (B) Samples were collected at 6, 12, and 24 h post-infection with SQ2014 (MOI = 0.1), and the protein levels of PEDV-N and actin were detected by Western blot. (C) After 12 h of infection, the protein samples with different concentrations of DNJ were detected by Western blot. (D) Quantitative detection of PEDV S and N mRNA level by qRT-PCR. (E) The cells were collected 12 h after infection, and the virus titer of SQ2014 was detected by PFU. The means and standard deviations of three independent experimental replicates are shown. ** p < 0.01; *** p < 0.001.

Article Snippet: The PEDV strain SQ2014 (GenBank accession No. KP728470) and Rabbit anti-PEDV N protein polyclonal antibodies were kindly provided by Professor Qian Yingjuan (Nanjing Agricultural University) [22]. βActin Rabbit polyclonal antibodies were purchased from Proteintech (20536-1-AP, Nanjing, China).

Techniques: Infection, Western Blot, Quantitative RT-PCR, Virus

Journal: Animals : an open access journal from MDPI

Article Title: Antiviral Activity of 1-Deoxynojirimycin Extracts of Mulberry Leaves Against Porcine Epidemic Diarrhea Virus.

doi: 10.3390/ani15091207

Figure Lengend Snippet: Figure 3. The inhibition stage of DNJ on PEDV. (A) This section describes the comprehensive approach for infecting Vero-E6 cells and administering DNJ at a concentration of 100 µM. The treatment procedure is divided into three separate phases: pre-treatment, co-treatment, and post- treatment. (B) In accordance with the treatment protocol depicted in (A), Western blot analysis was conducted to evaluate the protein levels of both the virus and actin. (C) DNJ directly targets PEDV SQ2014 experimental results.

Article Snippet: The PEDV strain SQ2014 (GenBank accession No. KP728470) and Rabbit anti-PEDV N protein polyclonal antibodies were kindly provided by Professor Qian Yingjuan (Nanjing Agricultural University) [22]. βActin Rabbit polyclonal antibodies were purchased from Proteintech (20536-1-AP, Nanjing, China).

Techniques: Inhibition, Concentration Assay, Western Blot, Virus

Journal: Cancers

Article Title: Partial Hepatectomy Promotes the Development of KRASG12V-Induced Hepatocellular Carcinoma in Zebrafish

doi: 10.3390/cancers16101793

Figure Lengend Snippet: The effect of s100a1 knockdown on the development of kras G12V -induced HCC in zebrafish larvae. ( A ) Experiment design of s100a1 knockdown and oncogene induction in Lipan larvae and kras+ larvae. ( B ) Western blot of S100A1with the total protein extracted from MO-injected zebrafish embryos at 24 hpf. β-actin served as a loading control. Each protein sample was pooled from 15 zebrafish embryos. The quantification of the intensity of S100A1normalized by the intensity of β-actin was at the lower panel. The uncropped image of blots is shown in . ( C ) Fluorescence images of morpholino-treated Lipan and kras+ larvae after 48 h of Dox treatment. ( D ) Measurement of liver size based on the fluorescence in ( B ) ( n ≥ 8). Circles, squares, and triangles indicate values of individual samples ( E ) Expression of sox5, sox9a, and sox9b in the kras+ livers at 24 h after PH/sham surgery as determined by RT-qPCR ( n = 3). Scale Bar = 200 μm. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: The effect of MO- s100a1 -ATG on S100A1 expression was validated in 1-dpf zebrafish embryos with western blotting using the Rabbit-anti-βactin antibody (bs-0061R, Bioss Antibodies, Woburn, MA, USA) (1:5000) and the Rabbit-anti-S100A1 antibody (50266-RP02; Sino Biological, Beijing, China) (1:1000).

Techniques: Knockdown, Western Blot, Injection, Control, Fluorescence, Expressing, Quantitative RT-PCR

Journal: The Journal of biological chemistry

Article Title: The well-developed actin cytoskeleton and Cthrc1 expression by actin-binding protein drebrin in myofibroblasts promote cardiac and hepatic fibrosis.

doi: 10.1016/j.jbc.2023.102934

Figure Lengend Snippet: Figure 2. Drebrin contributes to actin cytoskeleton formation in cardiac myofibroblasts. A, MA plot of genes differentially expressed between siCtrl and siDbn1 myofibroblasts. At 72 h after siRNA transfection, the cardiac myofibroblasts were lysed and subjected to RNA-Seq, and the MA plots were drawn using the TPM values obtained. The x-axis represents the TPM value for gene expression, and the y-axis represents fold change between the two groups. The green plots indicate the genes downregulated on silencing Dbn1 (M < −1.0, A > 0), and the red plots indicate the genes in GO terms associated with focal adhesion and regulation of the actin cytoskeleton. B, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of genes differentially expressed between the two groups. The genes downregulated on silencing Dbn1 (M < −1.0, A > 0) were analyzed using DAVID; the enriched KEGG pathways (gene count > 25) are shown. C, Acta2 or Tagln mRNA expression in cardiac myofibroblasts treated with siCtrl or siDbn1. At 72 h after siRNA transfection, the cardiac myofibroblasts were lysed and subjected to qRT-PCR. siCtrl: n = 5; siDbn1: n = 5. D, α-SMA, SM22α, or β-actin protein levels in cardiac myofibroblasts treated with siCtrl or siDbn1. At 60 h after siRNA transfection, the cardiac myofibroblasts were starved for 12 h, lysed, and subjected to immunoblot analysis. The panel below shows quantification of α-SMA, SM22α, or β-actin expression. E, phalloidin staining of F-actin in cardiac myo- fibroblasts treated with siCtrl or siDbn1. At 48 h after siRNA transfection, the cardiac myofibroblasts were seeded onto glass-bottom dishes, cultured for 24 h and subjected to phalloidin staining. Scale bar: 20 μm. The right panel shows quantification of phalloidin intensity. siCtrl: n = 32; siDbn1: n = 30. DAVID, Database for Annotation, Visualization and Integrated Discovery; TPM, transcript per million; α-SMA, α-smooth muscle actin.

Article Snippet: The antibodies used in this study were as follows: mouse anti-GAPDH antibody (1:20,000; Santa Cruz; sc-32233), mouse anti-α-SMA antibody (1:10,000; Thermo; MS-113-P), rabbit anti-SM22α antibody (1:4000; Abcam; ab14106), rabbit anti-Col1a1 antibody (1:4,000, CST; 72,026), rabbit anti-βactin antibody (1:4000; Proteintech; 20536-1-AP), rabbit antiMkl1 antibody (1:4000; Proteintech; 21166-1-AP), rabbit anti-lamin A/C antibody (1:4000; Proteintech; 10298-1-AP), rabbit anti-SOX9 antibody (1:4000; abcam; ab185230), horseradish peroxidase (HRP)-conjugated anti-FLAG primary antibody (1:10,000; Sigma Aldrich; A8592), horseradish peroxidase (HRP)- conjugated anti-HA primary antibody (1:5000; Roche; 12013819001), HRP-conjugated anti-mouse IgG secondary antibody (1:10,000; Abcam; ab6789), HRP-conjugated antirabbit IgG secondary antibody (1:10,000; Abcam; ab6721), HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000; Santa Cruz; sc-2004), and HRP-conjugated anti-rabbit IgG secondary antibody (1:2000; CST; 7074).

Techniques: Transfection, RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, Western Blot, Staining, Cell Culture

Journal: The Journal of biological chemistry

Article Title: The well-developed actin cytoskeleton and Cthrc1 expression by actin-binding protein drebrin in myofibroblasts promote cardiac and hepatic fibrosis.

doi: 10.1016/j.jbc.2023.102934

Figure Lengend Snippet: Figure 3. Drebrin promotes MRTF–SRF signaling. A, luminescence obtained using the SRF-RE reporter in drebrin-overexpressing NIH3T3 cells after CCG- 1423 treatment. At 24 h after plasmid transfection, the NIH3T3 cells were starved for 24 h, treated with CCG-1423 (10 μM) for 24 h, lysed, and subjected to qRT-PCR. n = 4. B, luminescence obtained using the SRF-RE reporter in Dbn1-knockdown NIH3T3 cells after TGF-β stimulation. At 48 h after siRNA trans- fection, the NIH3T3 cells were stimulated with TGF-β (2 ng/ml) for 24 h, lysed, and subjected to the luciferase assay. n = 4. C, Acta2 and Col1a1 mRNA expression levels in NIH3T3 cells treated with siCtrl or siDbn1 after TGF-β stimulation. At 48 h after siRNA transfection, the NIH3T3 cells were stimulated with TGF-β (2 ng/ml) for 24 h, lysed, and subjected to qRT-PCR. n = 4. D, binding between MRTF and α-SMA or β-actin in drebrin-overexpressing NIH3T3 cells. At 48 h after plasmid transfection, the NIH3T3 cells were lysed and subjected to the immunoprecipitation assay. n = 3. E, MRTF translocation in cardiac myofibroblasts treated with siCtrl or siDbn1. At 48 h after siRNA transfection, the cells were seeded onto glass-bottom dishes, cultured for 24 h, and subjected to immunocytochemistry. Scale bar: 20 μm. The right panel shows quantification of MRTF intensity. siCtrl; n = 15, siDbn1; n = 14. F, MRTF protein expressions in nuclear extraction of cardiac myofibroblasts treated with siCtrl or siDbn1. At 72 h after siRNA transfection, the cardiac myofibroblasts were lysed and subjected to immunoblot analysis. n = 3. G, Mkl1 or Mkl2 mRNA expression in cardiac myofibroblasts treated with siCtrl or siDbn1. At 72 h after siRNA transfection, the cardiac myofibroblasts were lysed and subjected to qRT-PCR. siCtrl: n = 5; siDbn1: n = 5. MRTF, myocardin-related transcription factor; SRF, serum response factor; α-SMA, α-smooth muscle actin; TGF, transforming growth factor.

Article Snippet: The antibodies used in this study were as follows: mouse anti-GAPDH antibody (1:20,000; Santa Cruz; sc-32233), mouse anti-α-SMA antibody (1:10,000; Thermo; MS-113-P), rabbit anti-SM22α antibody (1:4000; Abcam; ab14106), rabbit anti-Col1a1 antibody (1:4,000, CST; 72,026), rabbit anti-βactin antibody (1:4000; Proteintech; 20536-1-AP), rabbit antiMkl1 antibody (1:4000; Proteintech; 21166-1-AP), rabbit anti-lamin A/C antibody (1:4000; Proteintech; 10298-1-AP), rabbit anti-SOX9 antibody (1:4000; abcam; ab185230), horseradish peroxidase (HRP)-conjugated anti-FLAG primary antibody (1:10,000; Sigma Aldrich; A8592), horseradish peroxidase (HRP)- conjugated anti-HA primary antibody (1:5000; Roche; 12013819001), HRP-conjugated anti-mouse IgG secondary antibody (1:10,000; Abcam; ab6789), HRP-conjugated antirabbit IgG secondary antibody (1:10,000; Abcam; ab6721), HRP-conjugated anti-rabbit IgG secondary antibody (1:10,000; Santa Cruz; sc-2004), and HRP-conjugated anti-rabbit IgG secondary antibody (1:2000; CST; 7074).

Techniques: Plasmid Preparation, Transfection, Quantitative RT-PCR, Knockdown, Luciferase, Expressing, Binding Assay, Immunoprecipitation, Translocation Assay, Cell Culture, Immunocytochemistry, Extraction, Western Blot