stat3 luc reporter vector  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration

doi: 10.1016/j.jbc.2024.107793

Figure Lengend Snippet: Analysis of STAT3-dependent transcriptome and proteome changes in HAP-1 USP21 KO versus HAP-1 WT cells. Comparative transcriptomic and proteomic analysis of USP21 KO HAP-1 cells versus control cells. Volcano plots show differentially expressed genes ( A ) and proteins ( B ) in USP21 KO HAP-1 cells versus parental HAP-1 cells. The data points above the significance threshold (q < 0.05) are marked in red (upregulated in USP21 KO) and blue (downregulated in USP21 KO). The gray dashed vertical lines indicate the thresholds of |log2(FoldChange)| = 1 and |log2(FoldChange)| = 0.5 for transcriptomic and proteomic data, respectively. Transcripts and proteins with insignificant changes are depicted in gray . Transcription factors regulating expression of 20 most significantly downregulated transcripts and the most downregulated proteins were analyzed using ChIP Enrichment Analysis (ChEA) and Encode databases and the names are marked on volcano plots. Transcripts and proteins regulated by STAT3 are marked on volcano plots in green squares , regulated in STAT3-independent way are marked in gray squares . STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease

Article Snippet: pTF-STAT3-Luc reporter vector (BioCat) and pcDNA3.1-HA-USP21, or pcDNA3.1 plasmids were transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

Techniques: Control, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration

doi: 10.1016/j.jbc.2024.107793

Figure Lengend Snippet: USP21 is a positive regulator of STAT3 expression and phosphorylation in HAP-1 cells . The effect of USP21 KO ( A ) and overexpression ( B ) on STAT3 expression level and Tyr705 phosphorylation in HAP-1 cells was analyzed by Western blot and detection of pSTAT3, STAT3, USP21, and β-actin. A , HAP-1 cells (parental and USP21 KO, 1 × 10 6 /sample) were treated with 50 ng/ml IL-6 for up to 20 min. Then cell lysates were used for Western blot analysis. The histogram ( right panel ) depicts the levels of pSTAT3 in HAP-1 WT (red ) and HAP-1 USP21 KO ( blue ) determined through densitometry corrected for actin ( B ). For analysis of STAT3 phosphorylation in HAP-1 cells overexpressing USP21, cells were transfected with pcDNA3.1-USP21 vector (1 μg and 5 μg) by nucleofection. After 24 h cells were stimulated with IL-6 for 5 and 20 min. The level of pSTAT3, STAT-3, USP21, and β-actin was detected by Western blot. Histogram ( right panel ) represent relative mean band intensity of pSTAT3 for HAP-1 cells untransfected ( red ) and transfected with 1 μg ( blue ) and 5 μg ( gray ) of pcDNA3.1-USP21. The effect of USP21 overexpression on STAT3 activation analyzed by Dual-Glo luciferase system ( C ). pTF-STAT3-Luc, pcDNA3.1-HA-USP21, or pcDNA3.1 (EV; empty vector) plasmids were transfected in HEK293 cells for 8 h, followed by activation with IL-6 for 16 h. Then, the cells were subjected to luminescence measurement by using a Dual-Glo luminescence assay. Interaction of USP21 with STAT3 ( D ). HA-USP21 or STAT3 encoding plasmids were transfected into HEK293T for 48 h, and then cell lysates were prepared for coimmunoprecipitation and immunoblotting against specific antibodies as indicated. The standard deviation was determined from three independent repetitions. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns indicates no statistical difference ( p > 0.05). IL-6, interleukin 6; pSTAT3, Tyr705 phosphorylated signal transducer and activator of transcription 3; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Article Snippet: pTF-STAT3-Luc reporter vector (BioCat) and pcDNA3.1-HA-USP21, or pcDNA3.1 plasmids were transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

Techniques: Expressing, Over Expression, Western Blot, Transfection, Plasmid Preparation, Activation Assay, Luciferase, Luminescence Assay, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: The ubiquitin-specific protease 21 is critical for cancer cell mitochondrial function and regulates proliferation and migration

doi: 10.1016/j.jbc.2024.107793

Figure Lengend Snippet: USP21 drives proliferation, migration, and influences STAT3 phosphorylation in A549 cells . Cell proliferation of A549 transfected with scramble siRNA-siCONTROL ( red line ) and siUSP21 ( blue line ), was analyzed by cell counting every day for 4 days. The number of cells was determined automatically using Luna cell counter ( A ). The ability of cells in culture to grow was tested in the colony formation assay ( B ). A549 cells treated with scramble siRNA and siUSP21 were placed 2 × 10 2 cells in 24-well plates, incubated for 10 days, and the cells were stained with 0.1% crystal violet and counted ( left panel ). The mean number of colonies from six wells for scramble siRNA ( red ) and siUSP21 ( blue ) treated cells are presented on the histogram ( right panel ). A wound healing assay was used to assess the migration ability of A549 cells (the scale bar represents 200 μm) ( C ). Western blot analysis of STAT3 phosphorylation in IL-6-stimulated USP21 silencing A549 cells ( D ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. IL-6, interleukin 6; STAT3, signal transducer and activator of transcription 3; USP, ubiquitin-specific protease.

Article Snippet: pTF-STAT3-Luc reporter vector (BioCat) and pcDNA3.1-HA-USP21, or pcDNA3.1 plasmids were transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction.

Techniques: Migration, Transfection, Cell Counting, Colony Assay, Incubation, Staining, Wound Healing Assay, Western Blot

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: TFF1 expression alters H. pylori -induced transcriptional activation and regulation of NF-κB and STAT3 target genes. a-b Luciferase activity assay using NF-κB-Luc ( a ) and STAT3-Luc ( b ). AGS-pcDNA and AGS-TFF1 cells were co-transfected with NF-κB-Luc or STAT3-Luc and β-galactosidase plasmids. After 24 h, cells were infected with H. pylori 7.13 strain. Cells were collected 4 h after infection for NF-κB-Luc and 24 h for STAT3-Luc measurements. H. pylori 7.13 infection of AGS-pcDNA cells significantly increased the luciferase activity, which was reduced after the reconstitution of TFF1. The bar graphs represent the mean ± SEM of 3 independent experiments. c – f RT-qPCR analysis showing a decrease in mRNA expression of pro-inflammatory target genes ( IL-6,VEGF-α, IL-17 and IL-23 ) in AGS-TFF1 cells relative to AGS-pcDNA cells, following infection with H. pylori 7.13. The bars represent the mean ± SEM of three independent experiments

Article Snippet: To measure the transcription activity of NF-κB and STAT3 signal transduction pathway, we used the NF-κB-Luc (Cat# 631743) from (Clontech, Palo Alto, CA) and STAT3-Luc reporter vector (Cat# 8688) from (Addgene, Cambridge, MA).

Techniques: Expressing, Activation Assay, Luciferase, Activity Assay, Transfection, Infection, Quantitative RT-PCR

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: TFF1 attenuates H. pylori-induced activation of NF-κB and STAT3 in vitro. a–b ) Western blot analysis of STAT3 and NF-κB in AGS cell lines stably expressing TFF1 or empty vector pcDNA infected with H. pylori J166 ( a ) or 7.13 ( b ) strains at different time points 1, 6 and 24 h. H. pylori increases of P-NF-κB-P65 (S536) and P-STAT3 (Y705) protein levels after 1 and 24 h of infection, respectively and reconstitution of TFF1 expression abolished this increase. β-ACTIN was used as a protein loading control, and TFF1 expression was confirmed using TFF1 specific antibody. The relative intensity ratio of p-STAT3 (y705)/β-Actin and p-NF-κB/β-Actin were calculated by the Image-Lab software from BioRad. The Western blot results are representative of three independent experiments. The results are expressed as mean ± SEM of at least three independent experiments using a two-tailed Student’s t -test

Article Snippet: To measure the transcription activity of NF-κB and STAT3 signal transduction pathway, we used the NF-κB-Luc (Cat# 631743) from (Clontech, Palo Alto, CA) and STAT3-Luc reporter vector (Cat# 8688) from (Addgene, Cambridge, MA).

Techniques: Activation Assay, In Vitro, Western Blot, Stable Transfection, Expressing, Plasmid Preparation, Infection, Software, Two Tailed Test

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: TFF1 abolishes H. pylori -induced increase of NF-κB and STAT3 binding to its target gene IL6. a – b ChIP assay in AGS-pcDNA and AGS-TFF1 stable cells infected with H. pylori (7.13) for a period of 4 h for NF-κB binding ( a ) and 24 h for STAT3 binding ( b ), followed by quantitative real-time PCR with primers designed for STAT3 and NF-κB binding site on IL6 promoter regions. Control primers were designed 500 base pair upstream of STAT3 binding site on the IL6 promoter. These primers were used as negative control (NC). Results are presented as a percentage of input

Article Snippet: To measure the transcription activity of NF-κB and STAT3 signal transduction pathway, we used the NF-κB-Luc (Cat# 631743) from (Clontech, Palo Alto, CA) and STAT3-Luc reporter vector (Cat# 8688) from (Addgene, Cambridge, MA).

Techniques: Binding Assay, Infection, Real-time Polymerase Chain Reaction, Negative Control

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: BAY and Iκ-B super repressor inhibit STAT3 activation. a The luciferase reporter assay using a STAT3-Luc ( a ) reporter plasmids. H. pylori infection of AGS-pcDNA cells significantly increased the luciferase activity, which was reduced after treatment with BAY (10 µM) or transfection with Iκ-B super-repressor plasmid The bar graphs represent the mean ± SEM of 3 independent experiments. b Western blot analysis of p-STAT3 in AGS-pcDNA cell lines infected with H. pylori, 7.13 treated with BAY, or transfected with Iκ-B super repressor (Iκ-BSR). The increase of p-STAT (Y705) protein level in H. pylori -infected cells was abolished after treatment with BAY or transfected with Iκ-BSR plasmid. The results are representative of three independent experiments

Article Snippet: To measure the transcription activity of NF-κB and STAT3 signal transduction pathway, we used the NF-κB-Luc (Cat# 631743) from (Clontech, Palo Alto, CA) and STAT3-Luc reporter vector (Cat# 8688) from (Addgene, Cambridge, MA).

Techniques: Activation Assay, Luciferase, Reporter Assay, Infection, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Cancer Cell International

Article Title: NF-kB-dependent activation of STAT3 by H. pylori is suppressed by TFF1

doi: 10.1186/s12935-021-02140-2

Figure Lengend Snippet: Treatment with STAT3 or NF-κB inhibitors reduced inflammation in TFF1-KO mice gastric tissues. a Immunofluorescence staining of phospho-NF-κB-P65 (Green) and phospho-STAT3 (Y705) (Red) in the antropyloric region of the glandular stomach of the TFF1-KO mice infected with PMSS1 H. pylori and treated or not by intraperitoneal injection with BAY (5 mg/Kg). In control, un-infected TFF1-KO showed more p-NF-κB-P65 and p-STAT3 nuclear staining after H. pylori infection (arrowheads). However, after treatment with BAY, staining showed reduced nuclear STAT3 and NF-κB (arrows). 4’, 6’ Diamino-2-phenylindole (DAPI) (blue) was used as a nuclear counterstain, original magnification (× 1000). b RT-qPCR analysis showing mRNA expression of pro-inflammatory target genes ( Il-6, Vegf-α, Il-17 and IL-23 ) in H. pylori -infected TFF1-KO mice (10–12 weeks of age) treated or not with BAY (5 mg/Kg) and compared to TFF1-KO uninfected mice. Results are graphed using box-and-whisker blots to depict the smallest value, lower quartile, median, upper quartile, and largest value. (●) Indicate the mean. c A schematic cartoon depicting the role of TFF1 in regulating the inflammation in gastric epithelial cell through inhibition of NF-κB-mediated activation of STAT3 in response to H. pylori

Article Snippet: To measure the transcription activity of NF-κB and STAT3 signal transduction pathway, we used the NF-κB-Luc (Cat# 631743) from (Clontech, Palo Alto, CA) and STAT3-Luc reporter vector (Cat# 8688) from (Addgene, Cambridge, MA).

Techniques: Immunofluorescence, Staining, Infection, Injection, Quantitative RT-PCR, Expressing, Whisker Assay, Inhibition, Activation Assay