Journal: Cell Death Discovery

Article Title: N 6 -methyladenosine (m 6 A) methyltransferase KIAA1429 accelerates the gefitinib resistance of non-small-cell lung cancer

doi: 10.1038/s41420-021-00488-y

Figure Lengend Snippet: A Integrative genomics viewer (IGV) tool showed the remarkable m 6 A peak in the 3′-UTR of HOXA1 mRNA. B RT-qPCR was performed to detect the HOXA1 mRNA expression. C RNA-binding protein immunoprecipitation assay (RIP) demonstrated the interaction within KIAA1429 antibody (anti-KIAA1429) and m 6 A-modified HOXA1 mRNA. D MeRIP-qPCR with specific primers (HOXA1 mRNA primer) revealed the m 6 A modification level upon KIAA1429 knockdown or control. E KIAA1429-knockdown PC9-GR cells or control cells were treated with actinomycin D (1 μg/ml). The RNA decay rate was identified using qPCR normalized to initial 0-h expression. Bar graphs indicate the means ± SD. ** P < 0.01.

Article Snippet: For the transient transfection, siRNA targeting HOXA1 was designed and produced by RiboBio (Guangzhou, China) and transfected with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, RNA Binding Assay, Immunoprecipitation, Modification, Knockdown, Control

Journal: Cell Death Discovery

Article Title: N 6 -methyladenosine (m 6 A) methyltransferase KIAA1429 accelerates the gefitinib resistance of non-small-cell lung cancer

doi: 10.1038/s41420-021-00488-y

Figure Lengend Snippet: A HOXA1 expression was upregulated in the lung cancer cohort based on the TCGA database ( http://gepia.cancer-pku.cn ). B Gefitinib-resistant analysis was performed to detect the IC 50 value (gefitinib concentration causing 50% absorbance decreasing) in PC9-GR cells that transfected with KIAA1429 knockdown (sh-KIAA1429), HOXA1 overexpression (HOXA1 OV), HOXA1 knockdown (si-HOXA1), and KIAA1429 overexpression plasmid (KIAA1429 OV). C Wound-healing analysis was conducted using PC9-GR cells transfected with KIAA1429 knockdown (sh-KIAA1429), HOXA1 overexpression (HOXA1 OV), HOXA1 knockdown (si-HOXA1), and KIAA1429 overexpression plasmid (KIAA1429 OV). D Tumor weight and ( E ) volume were detected using PC9-GR cells transfected with KIAA1429 knockdown (sh-KIAA1429) and control (sh-NC). Bar graphs indicate the means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: For the transient transfection, siRNA targeting HOXA1 was designed and produced by RiboBio (Guangzhou, China) and transfected with Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay, Transfection, Knockdown, Over Expression, Plasmid Preparation, Control

Journal: Molecular Oncology

Article Title: Single‐cell RNA‐seq reveals the invasive trajectory and molecular cascades underlying glioblastoma progression

doi: 10.1002/1878-0261.12569

Figure Lengend Snippet: TFs and lncRNAs identified in data from Patel et al . . List of upregulated (A) and downregulated (B) TFs as well as their Spearman correlation coefficient with pseudotime. (C) Venn diagrams showed the significant overlaps of TFs between both GBM data sets. P values were calculated by hypergeometric test. (D) Expression profiles of the most positively correlated TF EPAS1 and the most negatively correlated TF OLIG1. Data points are fitted with local polynomial regression fitting (red lines) with 95% confidence interval (gray area). Cells are colored based on their states. List of the top 100 upregulated (E) and downregulated (F) lncRNAs as well as their Spearman correlation coefficient with pseudotime in GBM1.

Article Snippet: Short‐interfering RNAs (siRNAs) specifically against EPAS1 were purchased from RiboBio (Guangzhou, China) and then transfected into GBM cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Expressing

Journal: Molecular Oncology

Article Title: Single‐cell RNA‐seq reveals the invasive trajectory and molecular cascades underlying glioblastoma progression

doi: 10.1002/1878-0261.12569

Figure Lengend Snippet: Knockdown of EPAS1 inhibited GBM cell migration and invasion in vitro . (A) Endogenous EPAS1 expression status in three GBM cell lines. (B) EPAS1 expression was efficiently knocked down by two targeted siRNAs (siRNA1 and siRNA2) in U251 cells and LN229 cells as detected by Western blotting. Silencing EPAS1 expression suppressed cell migration and invasion capacity of U251 (C) and LN229 (D) cells in the Transwell migration and invasion assay (magnification 100×). Scale bars = 500 μm. Results were summarized as mean ± SD of three independent experiments (** P < 0.01; *** P < 0.001, independent Student’s t test).

Article Snippet: Short‐interfering RNAs (siRNAs) specifically against EPAS1 were purchased from RiboBio (Guangzhou, China) and then transfected into GBM cells using Lipofectamine 2000 reagent (Invitrogen, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Knockdown, Migration, In Vitro, Expressing, Western Blot, Invasion Assay

Journal: BMC Cardiovascular Disorders

Article Title: Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/β-catenin cascade

doi: 10.1186/s12872-024-03748-8

Figure Lengend Snippet: Mycn upregulation alleviates myocardial dysfunction in mice. Mice were administrated with lentiviral vectors-carried OE-Mycn or OE-NC, followed by ISO injection. A , HW/BW of mice. B , Myocardial injury of mice analyzed by HE staining. C , LVEF and LVFS or mice analyzed by echocardiographic assessment. D , Serum levels of CK, CK-MB, LDH, and cTnT in mice determined using ELISA kits. E , ROS content in the mouse heart determined using DHE staining. F - G , MDA content (F) and SOD activity (G) in the mouse heart tissue determined using ELISA kits. H , Concentrations of ANP and BNP in the mouse heart tissue determined using ELISA kits. I , Cell apoptosis in the mouse heart tissue examined using TUNEL assay. J , Mycn mRNA expression in the mouse heart tissue examined by RT-qPCR. In each group, n = 10. Differences were analyzed using the unpaired t test or the two-way ANOVA. * p < 0.05

Article Snippet: For artificial gene interference in mice, the animals were injected with lentiviral vectors-packaged gene overexpression plasmids of Mycn (OE-Mycn), USP2 (OE-USP2), gene knockdown plasmid of JUP (KD-JUP), or the negative control (NC) plasmids (OE-NC and KD-NC) (All provided by VectorBuilder Inc., Guangzhou, Guangdong, China).

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, TUNEL Assay, Expressing, Quantitative RT-PCR

Journal: BMC Cardiovascular Disorders

Article Title: Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/β-catenin cascade

doi: 10.1186/s12872-024-03748-8

Figure Lengend Snippet: Mycn activates USP2 transcription. A , USP2 predicted as a target of Mycn predicted in the hTFtarget system and the putative binding sites. B - C , mRNA (B) and protein (C) expression of USP2 in the heart tissue of mice after HCM modeling and OE-Mycn treatment examined by RT-qPCR and WB analysis, respectively. D , binding relationship between Mycn and the USP2 promoter in mouse cardiomyocytes determined by the ChIP assay. E , transcription activity of USP2 promoter in mouse cardiomyocytes transfected with OE-Mycn examined by the luciferase reporter gene assay. For animal experiments, n = 10 in each group. For cellular experiments, three biological replicates were conducted. Differences were analyzed by the unpaired t test or the one-way ANOVA. * p < 0.05. See full-length blot images in Supplementary Figure

Article Snippet: For artificial gene interference in mice, the animals were injected with lentiviral vectors-packaged gene overexpression plasmids of Mycn (OE-Mycn), USP2 (OE-USP2), gene knockdown plasmid of JUP (KD-JUP), or the negative control (NC) plasmids (OE-NC and KD-NC) (All provided by VectorBuilder Inc., Guangzhou, Guangdong, China).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Activity Assay, Transfection, Luciferase, Reporter Gene Assay

Journal: BMC Cardiovascular Disorders

Article Title: Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/β-catenin cascade

doi: 10.1186/s12872-024-03748-8

Figure Lengend Snippet: USP2 overexpression alleviates cardiac hypertrophy and dysfunction in mice. Mice were administrated with lentiviral vectors-carried OE-USP2 or OE-NC, followed by ISO injection. A , HW/BW of mice. B , Myocardial injury of mice analyzed by HE staining. C , LVEF and LVFS or mice analyzed by echocardiographic assessment. D , Serum levels of CK, CK-MB, LDH, and cTnT in mice determined using ELISA kits. E , ROS content in the mouse heart determined using DHE staining. F , SOD activity and MDA content in the mouse heart tissue determined using ELISA kits. G , Concentrations of ANP and BNP in the mouse heart tissue determined using ELISA kits. H , Cell apoptosis in the mouse heart tissue examined using TUNEL assay. I , Mycn mRNA expression in the mouse heart tissue examined by RT-qPCR. In each group, n = 10. Differences were analyzed using the unpaired t test or the two-way ANOVA. * p < 0.05

Article Snippet: For artificial gene interference in mice, the animals were injected with lentiviral vectors-packaged gene overexpression plasmids of Mycn (OE-Mycn), USP2 (OE-USP2), gene knockdown plasmid of JUP (KD-JUP), or the negative control (NC) plasmids (OE-NC and KD-NC) (All provided by VectorBuilder Inc., Guangzhou, Guangdong, China).

Techniques: Over Expression, Injection, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, TUNEL Assay, Expressing, Quantitative RT-PCR

Journal: BMC Cardiovascular Disorders

Article Title: Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/β-catenin cascade

doi: 10.1186/s12872-024-03748-8

Figure Lengend Snippet: Graphical abstract. In the context of cardiac hypertrophy, Mycn ameliorates heart failure by activating transcription of USP2, which enhances the stability of JUP protein via deubiquitination modification and subsequently blocks the Akt/β-catenin pathway

Article Snippet: For artificial gene interference in mice, the animals were injected with lentiviral vectors-packaged gene overexpression plasmids of Mycn (OE-Mycn), USP2 (OE-USP2), gene knockdown plasmid of JUP (KD-JUP), or the negative control (NC) plasmids (OE-NC and KD-NC) (All provided by VectorBuilder Inc., Guangzhou, Guangdong, China).

Techniques: Modification

Journal: Scientific Reports

Article Title: The modulation of MiR-155 and MiR-23a manipulates Klebsiella pneumoniae Adhesion on Human pulmonary Epithelial cells via Integrin α5β1 Signaling

doi: 10.1038/srep31918

Figure Lengend Snippet: ( A ) Schematic presentation of base pairing between the 3′ UTR of HMGN2 and miR-155 or miR-23a by erect likes. The mutant seed sequence of the HMGN2 3′ UTR matching miR-23a is also presented by dots. ( B ) Western blot analysis showing the change of HMGN2 protein level in K . pneumoniae infected A549 and HBE16 cells at different infection time (MOI = 100). ( C ) Western blot analysis showing the effect of miR-23a mimic on the protein expressions of HMGN2 in K . pneumoniae infected A549 or HBE16 cells (MOI = 100 for 2 hours, same as D – F ). ( D ) Western blot analysis showing the effect of miR-155 mimic or inhibitor on the protein expressions of HMGN2 in K . pneumoniae infected or uninfected cells. ( E ) A549 cells were co-transfected with miR-23a mimic or miR-NC along with HMGN2 3′ UTR wild-type (WT) or mutant (MUT) reporter. Luciferase activity was measured 24 hr after transfection. ( F ) The relative K . pneumoniae adhesion in A549 cells co-transfected with pEx-HMGN2 and miR-155 or miR-23a mimic. (Data are the mean ± SD and represent three individual experiments. **p < 0.01 compared with miR-NC and pEx-NC cotransfection).

Article Snippet: HMGN2 overexpression vector (pEx-HMGN2) containing CDS region of HMGN2 mRNA was cloned into pEx-GFP plasmid (GenePharma Inc. Shanghai, China) via EcoRI and BamHI restriction sites.

Techniques: Mutagenesis, Sequencing, Western Blot, Infection, Transfection, Luciferase, Activity Assay, Cotransfection

Journal: Scientific Reports

Article Title: The modulation of MiR-155 and MiR-23a manipulates Klebsiella pneumoniae Adhesion on Human pulmonary Epithelial cells via Integrin α5β1 Signaling

doi: 10.1038/srep31918

Figure Lengend Snippet: Western blot analysis showing the expressions of integrin α5, integrin β1 and F-actin ( A ) or Rac1-GTP and the total Rac1 ( B ) in A549 cells transfected with pEx-HMGN2 and/or siRNA-HMGN2 prior to K . pneumoniae exposure (MOI = 100 for 2 hours, same as C and D). The relative mRNA level of integrin α5 and β1 ( C ), the protein level of integrin α5, integrin β1 and Rac1-GTP ( D ) of A549 cells co-transfected with pEx-HMGN2 and miR-155 mimic prior to K . pneumoniae exposure. ( E ) The relative FN-cell adhesion of uninfected A549 cells transfected as ( C , D ). (Data are the mean ± SD and represent three individual experiments. **p < 0.01 normalized with miR-NC and pEx-NC co-transfection).

Article Snippet: HMGN2 overexpression vector (pEx-HMGN2) containing CDS region of HMGN2 mRNA was cloned into pEx-GFP plasmid (GenePharma Inc. Shanghai, China) via EcoRI and BamHI restriction sites.

Techniques: Western Blot, Transfection, Cotransfection

Journal: Scientific Reports

Article Title: The modulation of MiR-155 and MiR-23a manipulates Klebsiella pneumoniae Adhesion on Human pulmonary Epithelial cells via Integrin α5β1 Signaling

doi: 10.1038/srep31918

Figure Lengend Snippet: K . pneumoniae adhesion was measured on A549 cells transfected with miRNA mimics and then treated with RGD (50 nM, 24 hours) or NSC23766 (NSC, 50 μM, 2 hours) ( A ) or cytochalasin ( B ) (CytoB, 10 μM, 2 hours) ( C ) prior to K . pneumoniae exposure (MOI = 100 for 2 hours). Untreated miR-NC was defined as 100% of relative adhesion. ( B ) Western blot analysis showing the expressions of F-actin in A549 cells treated with RGD or NSC23766 under the same condition as ( A ). (Data are the mean ± SD and represent three individual experiments. **p < 0.01). ( D ) The schematic diagram depicting miR-155 and miR23a pathway: miRNAs promote intergrin α5β1 and Rac1 activities by targeting negative transcriptional regulators of integrin -HMGN2 and NFI, and result in actin polymerization (black solid lines). The exposure of K . pneumoniae causes the active reduction of these two miRNAs, which subsequently de-represses HMGN2 and NFI to inhibit integrin/Rac1 function and slows actin polymerization (green and red arrows).

Article Snippet: HMGN2 overexpression vector (pEx-HMGN2) containing CDS region of HMGN2 mRNA was cloned into pEx-GFP plasmid (GenePharma Inc. Shanghai, China) via EcoRI and BamHI restriction sites.

Techniques: Transfection, Western Blot

Journal: Biology Open

Article Title: Regulatory effects of electronic beam irradiation on mir-21/smad7-mediated collagen I synthesis in keloid-derived fibroblasts

doi: 10.1242/bio.018770

Figure Lengend Snippet: Electron beam irradiation decreased p38 phosphorylation and modulated mir-21/smad7 signaling. (A) qPCR results demonstrated the level of mir-21 was higher in keloid samples than normal skin samples, while the expression of smad7 was lower in keloid samples than normal skin samples. (B) A negative correlation between smad7 mRNA and mir-21 expression levels was observed. (C) Luciferase assay results revealed that mir-21 directly downregulated the expression of smad7 by binding to its 3′UTR. (D) Quantitative PCR data indicated that mir-21 levels were higher in KF compared with NF. KF and NF samples without electron beam irradiation were collected for this test. (E) qPCR data indicated that electron beam irradiation suppressed the expression of mir-21 in KF compared with NF. (F) qPCR data indicated that electron beam irradiation increased the mRNA level of smad7 in KF compared with NF, though it did not significantly affect the expression of p38. (G) Western blotting data indicated that electron beam irradiation increased the expression of smad7 and suppressed the phosphorylation of p38 in KF compared with NF. It did not affect the protein level of p38 in both KF and NF. Figure is representative of three experiments with similar results (normalized to NF group or without electronic beam treated group). Error bars in A-F indicate s.d.

Article Snippet: For smad7 knockdown treatment, cells (1.5×10 5 ) grown on 6-well plates were transfected with 100 pmol siRNA of smad7 (si-Smad7) or negative control (si-NC) using 8 μl siRNA-Mate transfection reagent (GenePharma, Shanghai, China).

Techniques: Irradiation, Phospho-proteomics, Expressing, Luciferase, Binding Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: Biology Open

Article Title: Regulatory effects of electronic beam irradiation on mir-21/smad7-mediated collagen I synthesis in keloid-derived fibroblasts

doi: 10.1242/bio.018770

Figure Lengend Snippet: Mir-21 modulates the phosphorylation of p38 by regulating smad7 at the protein level. (A,B) qPCR data revealed expression levels of mir-21 in NF (A) and KF (B) were elevated with mir-21 mimics or inhibitor transfection after 24 and 48 h. (C,D) qPCR results indicated that there were no significant changes in the mRNA level of smad7 after mir-21 mimics or inhibitor transfection in NF and KF. (E) Western blots indicated that mir-21 mimic transfection decreased the protein level of smad7 both in NF and KF; however, the transfection of mir-21 inhibitor improved the protein level of smad7 in KF and NF. (F) Western blots showed that mir-21 mimic transfection increased the protein level of Col-1 and phosphorylation of p38 in NF without changing the expression of p38. (G) Western blot showed that overexpression of smad7 in NF partly restored the regulatory effect of mir-21 mimics on smad7, Col-1 and p-p38 expression; while suppression of smad7 by siRNA also partly restored the regulation effect of mir-21 inhibitor on those protein expression. Figure is representative of three experiments with similar results (normalized to mimics NC or inhibitor NC). Error bars in A-D indicate s.d.

Article Snippet: For smad7 knockdown treatment, cells (1.5×10 5 ) grown on 6-well plates were transfected with 100 pmol siRNA of smad7 (si-Smad7) or negative control (si-NC) using 8 μl siRNA-Mate transfection reagent (GenePharma, Shanghai, China).

Techniques: Phospho-proteomics, Expressing, Transfection, Western Blot, Over Expression

Journal: Biology Open

Article Title: Regulatory effects of electronic beam irradiation on mir-21/smad7-mediated collagen I synthesis in keloid-derived fibroblasts

doi: 10.1242/bio.018770

Figure Lengend Snippet: Electronic beam irradiation mediated Col-1 synthesis via mir-21/smad7 pathway in keloid-derived fibroblasts. (A) The expression level of mir-21 was downregulated by electronic beam irradiation; while mir-21 mimic transfection restored the inhibitory effect of electronic beam irradiation on mir-21 expression. (B) The expression levels of smad7 and p38 protein were promoted while the expression levels of Col-1 and p-p38 protein were reduced by the electronic beam irradiation; mir-21 mimic transfection could restore the regulatory effect of the electronic beam irradiation on protein expression of smad7, p38, Col-1 and p-p38. Figure is representative of three experiments with similar results (normalized to mimics NC or without electronic beam treated group). Error bars in A indicate s.d.

Article Snippet: For smad7 knockdown treatment, cells (1.5×10 5 ) grown on 6-well plates were transfected with 100 pmol siRNA of smad7 (si-Smad7) or negative control (si-NC) using 8 μl siRNA-Mate transfection reagent (GenePharma, Shanghai, China).

Techniques: Irradiation, Derivative Assay, Expressing, Transfection

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: BoHV-1 infection has no obvious effects on NFAT5 steady-state protein levels at late stages after infection. (A) Confluent MDBK cells in 60 mm dishes were infected with BoHV-1 at an MOI of 0.1 for 16 h and 24 h (h). Cells were lysed with RIPA buffer and then analyzed by Western blotting to detect NFAT5 protein levels. β-Actin was detected as a loading control. (B) Band intensity was analyzed with free software Image J. Each analysis was compared with that of mock-infected controls at indicated times after infection, which was arbitrarily set as 1. (C) MDBK cells in 6-well plates were either mock-infected or infected with BoHV-1 at an MOI of 0.1. At 24 h postinfection, the total RNA was purified, and the mRNA levels of NFAT5 were examined with qRT-PCR. The results shown are representations of three independent experiments, with error bars indicating standard deviations. Significance was assessed with a Student's t test ( *, P < 0.05; ns, not significant).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Infection, Western Blot, Software, Purification, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of BoHV-1 infection had on NFAT5 localization. MDBK cells in a 2-well chamber slide were either mock-infected or infected with BoHV-1 at an MOI of 0.1. After infection for 24 h, the cells were fixed with 4% formaldehyde, and NFAT5 protein was immunostained by using NFAT5-specific MAb via IFA. DAPI staining was used to stain nuclear DNA. Images were obtained by performing confocal microscopy (Leica).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Infection, Staining, Confocal Microscopy

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of the BoHV-1 infection had on the accumulation of NFAT5 in mitochondria. (A) MDBK cells in 100 mm dishes were either mock-infected or infected with BoHV-1 (MOI = 0.1) for 24 h. The cells were collected for isolation of mitochondria fractions via using a commercial kit (Beyotime Biotechnology, cat# C3601) following the manufacturer’s protocol. Protein levels of NFAT5 were detected by Western blotting. HSP60 was detected and used as a protein loading control. (B) Band intensity was analyzed with the software Image J. The control was arbitrarily set as 1. The results shown are representations of three independent experiments. Error bars denote the variability between three independent experiments. Significance was assessed with a Student's t test (*, P < 0.05).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Infection, Isolation, Western Blot, Software

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of the BoHV-1 infection had on nuclear accumulation of NFAT5 proteins. (A) MDBK cells in 100 mm dishes were either mock-infected or infected with BoHV-1 (MOI = 0.1) for 24 h. The cells were collected for isolation of both nucleus and cytosol fractions via using a commercial kit (Beyotime Biotechnology, cat# P0027), following the manufacturer’s protocol. Protein levels of NFAT5 were detected by Western blotting. LaminA/C, a marker for nuclear protein, and β-tubulin, a marker for cytoplasmic protein, were detected as a protein loading control, respectively. The results shown are representations of three independent experiments.

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Infection, Isolation, Western Blot, Marker

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of the BoHV-1 infection had on the transcription of NFAT5 downstream targets. MDBK cells in 6-well plates were either mock-infected or infected with BoHV-1 at an MOI of 0.1. At 24 h postinfection, total RNA was purified, and the mRNA levels of PGK1 (A), SMIT (B), and BGT-1 (C) were examined with qRT-PCR. The results shown are representations of three independent experiments, with error bars indicating standard deviations. Significance was assessed with a Student's t test ( *, P < 0.05).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Infection, Purification, Quantitative RT-PCR

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: NFAT5-specific siRNA increases virus production in MDBK cells. (A) MDBK cells in 6-well plates were transfected with either scrambled siRNA (200 pmol) or three individual siRNA targeting NFAT5 (200 pmol), referred to as siRNANFAT5-1, siRNANFAT5-2, and siRNANFAT5-3, respectively. At 48 h after transfection, the effective siRNA were screened by detection of NFAT5 protein levels via Western blot. (B) Band intensity was analyzed with the software Image J. The control was arbitrarily set as 100%. (C and D) MDBK cells in six-well plates were transfected with either scrambled siRNA (200 pmol) or siRNANFAT5-3 (200 pmol). After transfection for 48 h, cell cultures were infected with BoHV-1 (MOI = 0.1). After infection for 24 h, the cellular genome was purified by using a commercial viral DNA purification kit, and the viral genome was examined by using qPCR using gC-specific primer(C). In parallel, cell infection virus yield in supernatants was examined with results expressed as TCID 50 /mL (D). (E and F) NFAT5 plasmid of 1 and 2.5 μg along with empty vector was transfected into MDBK cells in 6-well plates using, after transfection for 48 h, the cells were either collected for the detection of NFAT5 protein levels using Western blot (E), or subjected to virus infection (MOI = 0.1), and the virus production were tittered in MDBK cells with results expressed as TCID 50 /mL (F). The data represented three independent experiments. Results are the mean of three independent experiments, with error bars showing standard deviations. Significance was assessed with a Student's t test (*, P < 0.05; ns, not significant).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Transfection, Western Blot, Software, Infection, Purification, DNA Purification, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of NFAT5-specific inhibitor KRN5 had on BoHV-1 productive infection. (A) MDBK cells in 24-well plates were treated with either DMSO control or 10 μM KRN5. After treatment for 24 h, the cytotoxicity in MDBK cells was analyzed with a Trypan-blue exclusion test. (B) Confluent MDBK cells in 24-well plates were pretreated with either DMSO control or KRN5 at a concentration ranging from 0.5 to 10 μM at 2 h before infection. The cells were infected with BoHV-1 at MOI of 0.1 for 1 h in the presence of either DMSO or KRN5. After extensive washing with PBS, a fresh medium containing inhibitors or DMSO control was replaced. At 12 and 24 h postinfection, the cell cultures were collected, and virus titers were measured with results expressed as TCID 50 /mL. (C to E) As described in panel B, MDBK were treated with 2 μM KRN5 during virus infection plus a pretreatment for 2 h prior to infection. After infection for 24 h, total RNA was isolated and analyzed by qRT-PCR to measure viral mRNA transcripts of bICP0 (C), DNA polymerase (D), and gC (E) by using relative qRT-PCR. (F) As described in panels C to E, MDBK cells in 6-well plates were either mock infected or infected with BoHV-1 along with treatment of either DMSO or 2 μM KRN5 during virus infection. After infection for 24 h, cell lysates were prepared and subjected to Western blot to detect viral protein gC. Data represent the means of three independent experiments. Significance was assessed with Student's t test (*, P < 0.05).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Infection, Concentration Assay, Isolation, Quantitative RT-PCR, Western Blot

Journal: Microbiology Spectrum

Article Title: NFAT5 Restricts Bovine Herpesvirus 1 Productive Infection in MDBK Cell Cultures

doi: 10.1128/spectrum.00117-23

Figure Lengend Snippet: Effects of KRN5 had on NFAT5 protein expression in MDBK cells. (A) MDBK cells in 6-well plates were treated with either DMSO control or KRN5 at indicated concentrations. After treatment for 24 h, the cell lysates were prepared and subjected to Western blot to detect the protein levels of NFAT5. (B) Band intensity was analyzed with the software Image J. The control was arbitrarily set as 100%. Data represent the means of three independent experiments. Significance was assessed with Student's t test (ns, not significant).

Article Snippet: MDBK cells in 6-well plates were transfected with scrambled siRNA or three NFAT5 specific siRNA of 200 pmol, provided by Genepharma (Shanghai, China) and plasmid pEGFP-NFAT5 (no EGFP), by using Lipofectamine 3000 (Invitrogen).

Techniques: Expressing, Western Blot, Software