Journal: Journal of cellular biochemistry

Article Title: Nuclear factor of activated T cells 2 is required for osteoclast differentiation and function in vitro but not in vivo.

doi: 10.1002/jcb.27212

Figure Lengend Snippet: FIGURE 5 Acp5, Ctsk, and Calcr mRNA levels are decreased in Lyz2Cre;Nfatc2Δ/Δ osteoclasts. BMMs derived from 1‐month old Lyz2Cre/WT;Nfatc2Δ/Δ mice and Nfatc2loxP/loxP control littermates were cultured for 4 days in the presence of M‐CSF at 30 ng/mL and of RANKL at 10 ng/mL. The cells were collected at the indicated times for extraction of total RNA and proteins. (A) Nfatc2 and Nfatc1 mRNA levels were measured by qRT‐PCR. Transcript levels are reported as copy number corrected for Rpl38 mRNA levels. Values are means ± SD; n = 5 control and n = 3 Nfatc2Δ/Δ biological replicates. Two technical replicates were used for each qRT‐PCR reaction. *Significantly different between Nfatc2Δ/Δ and control, P < 0.05. (B) 50 μg of total protein were separated by SDS‐PAGE and NFATc1 and NFATc2 levels were detected by using anti‐NFATc1 and anti‐NFATc2 antibodies, respectively. β‐Actin levels served as a loading control in the same blot. (C) Tnfrs11a, Acp5, Ctsk, and Calcr mRNA levels in total RNA were measured by qRT‐PCR. Transcript levels are reported as copy number corrected for Rpl38. Values are means ± SD; n = 5 control and n = 3 Nfatc2Δ/Δ biological replicates. Two technical replicates were used for each qRT‐PCR reaction. *Significantly different between Nfatc2Δ/Δ and control, P < 0.05. BMM, bone marrow‐derived macrophage; M‐CSF, macrophage‐colony stimulating factor; NFAT, nuclear factor of activated T cells; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; qRT‐PCR, quantitative reverse transcription‐PCR; SDS, sodium dodecyl sulfate

Article Snippet: Transcript copy number was estimated by comparison with a serial dilution of cDNA for Nfatc1 (Addgene plasmid 11793), Nfatc2 (Addgene plasmid 11791, both created by A Rao, La Jolla, CA), Acp5, Ctsk, and Calcr (Thermo Fisher Scientific).

Techniques: Derivative Assay, Control, Cell Culture, Extraction, Quantitative RT-PCR, SDS Page, Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Reverse Transcription

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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Western Blot, Control, 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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Isolation, Western Blot, Control, 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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Isolation, Western Blot, Marker, Control

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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Virus, Transfection, Western Blot, Software, Control, 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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Infection, Control, Concentration Assay, Virus, 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: The following antibodies were used in this study: NFAT5 monoclonal antibody (MAb) (cat# sc-398171) and LaminA/C mouse MAb (cat# sc-376248) were provided by Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Control, Western Blot, Software

Journal: Cell reports

Article Title: A multiplexed in vivo approach to identify driver genes in small cell lung cancer

doi: 10.1016/j.celrep.2023.111990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following antibodies were used: anti-HSP90 (CST 4877, 1:4,000), anti-TSC1 (D43E2, CST 6935, 1:200), anti-TSC2 (D93F12, CST 4308), anti-S6 (CST 2217, 1:200), anti-phospho-S6 (Ser235/236, CST 2211, 1:200), anti-GFP (D5.1, CST 2956, 1:200), anti-p-mTOR (D9C2, Ser2448, CST 55365, 1:200), anti-mTOR (CST 2972, 1:200), anti-phospho-4E-BP1 (Ser65, CST 9451, 1:200), and anti-4E-BP1 (53H11, CST 9644, 1:200).

Techniques: Plasmid Preparation, Virus, Recombinant, Modification, Labeling, Amplification, Polymer, Staining, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Simple Western, Membrane, DNA Extraction, Purification, Sequencing, Generated, Software