Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a qRT-PCR analysis of miR-4293 levels in NSCLC tissues and corresponding para-carcinoma tissues ( n = 16). Data are expressed as median (interquartile range). ** P < 0.01; Mann–Whitney U test. b qRT-PCR analysis of miR-4293 levels in A549, H1299, H1975, and HBE cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. c qRT-PCR analysis of miR-4293 level in A549 cells with transfection of miR-4293 mimic, ASO-4293, mu-4293 (mutant), or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. d MTT assay of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293 or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01, * p < 0.05; ANOVA test. e FACS analysis of A549 cell apoptosis at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. f Western blot detection of STAT3 and apoptosis-related factors (Bcl-2, Bax, BIRC3, and Caspase 3,8,9) in miR-4293-, ASO-4293-, or scramble-treated A549 cells. g , h Transwell migration assay of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. i , j Colony formation assays of A549 at 24 h post-transfection of miR-4293, ASO-4293, mu-4293, or scrambled. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Quantitative RT-PCR, MANN-WHITNEY, Transfection, Mutagenesis, MTT Assay, Western Blot, Transwell Migration Assay

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a TargetScan analysis predicted that the DCP2 mRNA-3′-UTR is targeted by miR-4293. We synthesized a mutant miR-4293 (Mu-4293) and cloned DCP2 mRNA-3’-UTR and mu-DCP2–3’-UTR sequences. b Analysis of miR-4293 regulating luciferase expression of reporter plasmids containing DC2–3’UTR (right panel) or mu-DCP2–3’UTR (left panel). Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. c Analysis of ASO-4293 regulating luciferase expression of reporter plasmids containing DC2–3’UTR (right panel) or mu-DCP2–3’UTR (left panel). Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. d Western blot detection of DCP2 in miR-4293-, ASO-4293-, or scramble-treated A549 cells. e Western blot detection of DCP2 in siRNA-DCP2–1-, siRNA-DCP2–2-, or scramble-treated A549 cells and H129 cells. f qRT-PCR analysis of WFDC21P level in pcDNA-WFDC21P + scramble-, si-DCP2-, si-DCP2 + pcDNA-WFDC21P-, or scramble-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. g Western blot detection of STAT3 phosphorylation in pcDNA-WFDC21P + scramble-, si-DCP2-, si-DCP2 + pcDNA-WFDC21P-, or scramble-treated A549 cells. h qRT-PCR analysis of DCP2 level in pcDNA or pcDNA-DCP2 -treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. i qRT-PCR analysis of WFDC21P level in 0 h, 4 h, 8 h, 12 h, 24 h, 48 h after transfection of pcDNA-DCP2. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. j RIP assay of the interaction between WFDC21P and DCP2. Upper panel, qRT-PCR analysis of WFDC21P pull-down by IgG or DCP2; lower panel, electrophoresis for WFDC21P pull-down by IgG or DCP2 after PCR amplification. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. k qRT-PCR analysis of WFDC21P levels in miR-4293-, ASO-4293, Mu-4293 or scramble-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. l qRT-PCR analysis of WFDC21P levels in miR-4293+pcDNA-WFDC21P-, or scramble+ pcDNA-WFDC21P-treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. m Western blot detection of STAT3 phosphorylation in pcDNA-WFDC21P + scramble-, miR-4293-, miR-4293+pcDNA-WFDC21P-, or scramble-treated A549 cells. n Western blot detection of STAT3 phosphorylation in AG490 + pcDNA-, pcDNA-WFDC21P-, AG490 + pcDNA-WFDC21P-, or pcDNA treated A549 cells. o qRT-PCR analysis of miR-4293 level in AG490 + pcDNA-, pcDNA-WFDC21P-, AG490 + pcDNA-WFDC21P-, or pcDNA treated A549 cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01, * P < 0.05; ANOVA test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Synthesized, Mutagenesis, Clone Assay, Luciferase, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Electrophoresis, Amplification

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a qRT-PCR analysis for WFDC21P level in NSCLC tissues and corresponding para-carcinoma tissues ( n = 16). Data are expressed as median (interquartile range). ** P < 0.01; Mann–Whitney U test. b qRT-PCR analysis for WFDC21P level in A549, H1299, H1975, and HBE cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. c Western blot detection of STAT3 in A549, H1299, H1975, and HBE cells. d qRT-PCR analysis of STAT3 expression in NSCLC tissues and corresponding para-carcinoma tissues ( n = 16). Data are expressed as median (interquartile range). ** P < 0.01; Mann–Whitney U test. e – g Pearson′s correlation analysis of miR-4293 expression and WFDC21P expression ( n = 16, ** P = 0.015) ( e ), miR-4293 expression and STAT3 expression (n = 16, ** P < 0.01) ( f ), WFDC21P expression and STAT3 expression ( n = 16, ** P < 0.01) ( g ).

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: a MTT assays of A549 at 24 h post-transfection of pcDNA-WFDC21P and pc-DNA. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. b qRT-PCR analysis of WFDC21P level in si-WFDC21P- and scramble-treated cells. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. c MTT assays of A549 at 24 h post-transfection of si-WFDC21P and scramble. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. d FACS analysis of A549 cell apoptosis at 24 h post-transfection of pc-DNA, pcDNA-WFDC21P, si-WFDC21P, and scrambled control. e Western blot detection of STAT3 and apoptosis-related factors (Bcl-2, Bax, BIRC3, and Caspase 3,8,9) pcDNA vs pcDNA-WFDC21P; scramble vs si-WFDC. f – i Transwell assays of A549 migrat i on. f , g pcDNA vs pcDNA-WFDC21P; ( h ) and ( i ) scramble vs. si-WFDC. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test. j – m Colony formation assays of A549. j , k pcDNA vs pcDNA-WFDC21P; ( l ) and ( m ) scramble vs. si-WFDC. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; Student’s t -test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: The above-mentioned data showed that miR-4293 transfection elevated miR-4293 levels, which could be attenuated by ASO. pcDNA-WFDC21P transfection increased WFDC21P. Si-WFDC treatment reduced WFDC21P level. a MTT assay of A549 at 24 h post-transfection of si-WFDC + scramble, miR-4293, miR-4293+si-WFDC, and scramble. Data are expressed as mean ± SD for triplicate experiments. ** p < 0.01; ANOVA test. b FACS analysis of A549 cell apoptosis at 24 h post-transfection of si-WFDC + scramble, miR-4293, miR-4293+si-WFDC, and scramble. c Western blot detection p-STAT3, Bcl-2, BAX, cleaved-caspase 3, 8, and 9 in si-WFDC + scramble-, miR-4293-, miR-4293+si-WFDC- or scramble-treated cells. d , e RTCA station analysis of migration for A549 with transcription of si-WFDC + scramble-, miR-4293-, miR-4293+si-WFDC- or scramble. f MTT of A549 at 24 h post-transfection of pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble. Data are expressed as mean ± SD for triplicate experiments. (* P < 0.05). g FACS analysis of A549 cell apoptosis at 24 h post-transfection of pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble. h Western blot detection of STAT3 phosphorylation and apoptosis-related factors (Bcl-2, Bax and Caspase 3) in pcDNA+ASO-4293, pcDNA-WFDC21P + scramble, pcDNA-WFDC21P + ASO-4293, and pcDNA+ scramble-treated A549 cells. i – l RTCA station analysis of migration for A549. i , j pcDNA-WFDC21P vs pc-DNA; ( k ) and ( l ), pcDNA-WFDC21P + scramble vs. pcDNA-WFDC21P + ASO-4293. Data are expressed as mean ± SD for triplicate experiments. * p < 0.05, *** p < 0.001; Student’s t -test.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Transfection, MTT Assay, Western Blot, Migration

Journal: Cell Death & Disease

Article Title: miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

doi: 10.1038/s41419-021-04021-y

Figure Lengend Snippet: miR-4293 promotes tumor cell proliferation and metastasis but suppresses apoptosis. DCP2 can directly or indirectly bind to and downregulate WFDC21P. By targeting DCP2 mRNA, miR-4293 suppresses the expression of DCP2, and further enhances the expression of WFDC21P and promotes STAT3 phosphorylation. As a result of elevated STAT3 activity, apoptosis is suppressed and proliferation is enhanced through Bcl2 elevation, Bax2 attenuation and reduced caspase cleavage. In addition, WFDC21P elevates miR-4293 expression by promoting STAT3 activation.

Article Snippet: The antibodies used were as follows: rabbit anti-human DCP2 (1:1000; ab28658; abcam, Ltd), rabbit anti-human STAT3 (1:500; bsm-33218M; Bioss, Ltd), rabbit antihuman p-STAT3 (1:800; AP0248), mouse anti-human BIRC3 (1:500; MB0129), rabbit anti-human Bax (1:500; BS6420), Bcl2 (1:500; BS70205), rabbit anti-human Caspase 3 (1:1000; BS61583), rabbit anti-human Caspase 8(1:1000; AP0258), rabbit anti-human Caspase 9 (1:1000; AP0359), and rabbit anti-human GAPDH (1:3000; MB001) all from Bioworld Technology, Ltd).

Techniques: Expressing, Activity Assay, Activation Assay

Journal: Journal of Immunology Research

Article Title: TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38 −/− Sepsis Mice

doi: 10.1155/2021/6687555

Figure Lengend Snippet: Escherichia coli can induce liver injury in mice. WT mice were injected with PBS or 3 × 10 8 cfu/ml E. coli intraperitoneally and sacrificed 3 hours later. Liver pathological injuries were observed with hematoxylin and eosin staining (a–d). The yellow arrows indicate edema, the blue arrows indicate inflammatory cell infiltration, the black arrows indicate punctate necrosis, and the green arrows indicate binucleate hepatocytes. Bacteria from the liver of PBS- or E. coli -stimulated WT mice were cultured in MH medium (e, f) overnight and identified by Gram staining (g). The serum AST (h) and ALT (i) concentrations were determined by using the detection kits. The mRNA of liver inflammatory cytokines TLR4 (j), NLRP3 (k), IL-1 β (l), and IL-18 (m) of PBS- or 3 × 10 8 cfu/ml E. coli -stimulated mice were measured by RT-qPCR. Data are presented as means ± standard deviation. Statistical significance was determined by the paired t -test ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The primary antibodies anti-TLR4 rabbit mAb (1 : 500) (CST, USA), anti-TRIF rabbit mAb (1 : 1000) (Proteintech, USA), anti-MyD88 mouse mAb (1 : 2000) (Proteintech, USA), anti-NF- κ B p65 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-NF- κ B p65 rabbit mAb (1 : 500) (CST, USA), anti-IL-6 mouse mAb (1 : 2000) (Proteintech, USA), anti-iNOS rabbit mAb (1 : 1000) (CST, USA), anti-BAX rabbit mAb (1 : 5000) (Proteintech, USA), anti-NLRP3 rabbit mAb (1 : 500) (Boster, China), anti-ASC rabbit mAb (1 : 500) (Affinity, China), anti-caspase-1 rabbit mAb (1 : 500) (Abcam, UK), anti-IL-1 β rabbit mAb (1 : 1000) (CST, USA), anti-IL-18 rabbit mAb (1 : 1000) (Affinity, China), anti-caspase-3 rabbit mAb (1 : 500) (CST, USA), anti-GSDMD rabbit mAb (1 : 500) (Affinity, China), anti-ERK1/2 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-ERK1/2 rabbit mAb (1 : 2000) (CST, USA), and anti-GAPDH rabbit mAb (1 : 5000) (Proteintech, USA) were used.

Techniques: Injection, Staining, Bacteria, Cell Culture, Quantitative RT-PCR, Standard Deviation

Journal: Journal of Immunology Research

Article Title: TLR4-NLRP3-GSDMD-Mediated Pyroptosis Plays an Important Role in Aggravated Liver Injury of CD38 −/− Sepsis Mice

doi: 10.1155/2021/6687555

Figure Lengend Snippet: The expression levels of pyroptosis-related markers were detected by Western blot. The expressions of liver pyroptosis proteins in WT, CD38 −/− , and CD38 −/− TLR4 mut mice were detected at 3 hours after E. coli stimulation by Western blot. The expressions of NLRP3, ASC, procaspase-1, cleaved caspase-1, IL-1 β , IL-18, procaspase-3, and cleaved caspase-3 were measured (a). And relative levels of NLRP3 to GAPDH (b), ASC to GAPDH (c), cleaved to procaspase-1 (d), IL-1 β to GAPDH (e), IL-18 to GAPDH (f), and cleaved to procaspase-3 (g) were analyzed by ImageJ software. Data are presented as means ± standard deviation. Statistical significance was determined by one-way ANOVA ( n = 3, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: The primary antibodies anti-TLR4 rabbit mAb (1 : 500) (CST, USA), anti-TRIF rabbit mAb (1 : 1000) (Proteintech, USA), anti-MyD88 mouse mAb (1 : 2000) (Proteintech, USA), anti-NF- κ B p65 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-NF- κ B p65 rabbit mAb (1 : 500) (CST, USA), anti-IL-6 mouse mAb (1 : 2000) (Proteintech, USA), anti-iNOS rabbit mAb (1 : 1000) (CST, USA), anti-BAX rabbit mAb (1 : 5000) (Proteintech, USA), anti-NLRP3 rabbit mAb (1 : 500) (Boster, China), anti-ASC rabbit mAb (1 : 500) (Affinity, China), anti-caspase-1 rabbit mAb (1 : 500) (Abcam, UK), anti-IL-1 β rabbit mAb (1 : 1000) (CST, USA), anti-IL-18 rabbit mAb (1 : 1000) (Affinity, China), anti-caspase-3 rabbit mAb (1 : 500) (CST, USA), anti-GSDMD rabbit mAb (1 : 500) (Affinity, China), anti-ERK1/2 rabbit mAb (1 : 1000) (CST, USA), anti-phospho-ERK1/2 rabbit mAb (1 : 2000) (CST, USA), and anti-GAPDH rabbit mAb (1 : 5000) (Proteintech, USA) were used.

Techniques: Expressing, Western Blot, Software, Standard Deviation

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

doi: 10.3389/fcimb.2017.00140

Figure Lengend Snippet: Oligonucleotide sequences used in the quantitative reverse transcription polymerase chain reaction (qRT-PCR) .

Article Snippet: For intracellular staining, a cytofix/cytoperm kit was used (Becton Dickinson) with fluorescently labeled antibodies against transcription factors: Alexa Fluor®488 Mouse Anti-Stat3 (pY705; BD Biosciences) and PE-IRF4 Monoclonal Antibody (3E4)(eBioscience).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

doi: 10.3389/fcimb.2017.00140

Figure Lengend Snippet: Activation of Th17 signaling pathway in CD4+ naïve lymphocyte depends on gingipain activity . Monocyte-derived dendritic cells (moDC) were untreated or exposed to P. gingivalis in the presence or absence of specific protease inhibitors (KYT-1 and KYT-36, each at a concentration of 1 μM). After 6 h, CD4+ naïve lymphocytes were added and co-stimulated for 3 consecutive days. At day 1, 2, and 3 after co-incubation, cells were collected and lysed with TRIzol, RNA was isolated and reverse transcriptase PCR was performed. Relative expression of cytokine genes IL-6R, IL-1R, TGF β R, RORc, ROR α , STAT3, RUNX1, IRF4, BATF to the reference house-keeping gene EF2 was measured by Real-Time PCR. Data represent fold increase in expression compared to control levels, which were arbitrarily set at 1 and were analyzed with a Student's t -test ( # P < 0.05, ## P < 0.01, ### P < 0.001 vs. control, * P < 0.05, ** P < 0.01, *** P < 0.001 to P. gingivalis treated cells).

Article Snippet: For intracellular staining, a cytofix/cytoperm kit was used (Becton Dickinson) with fluorescently labeled antibodies against transcription factors: Alexa Fluor®488 Mouse Anti-Stat3 (pY705; BD Biosciences) and PE-IRF4 Monoclonal Antibody (3E4)(eBioscience).

Techniques: Activation Assay, Activity Assay, Derivative Assay, Concentration Assay, Incubation, Isolation, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Inactive Gingipains from P. gingivalis Selectively Skews T Cells toward a Th17 Phenotype in an IL-6 Dependent Manner

doi: 10.3389/fcimb.2017.00140

Figure Lengend Snippet: Activation of STAT3 and IRF4 is dependent on gingipain activity . At the end of each period, T cells were stimulated with PMA and ionomycin for 4 h in the presence of GolgiStop. Cells were stained for intracellular (A) IRF4 and (B) pSTAT3 (pY705), and IRF4, and analyzed by flow cytometry. Data show fold increase of the transcription factor positive population, relative to the appropriate control set at 1. Data are presented as a mean ± standard deviation of triplicate assays and were analyzed with a Student's t -test ( # P < 0.05, ## P < 0.01, ### P < 0.001 vs. control, * P < 0.05, ** P < 0.01, *** P < 0.001 to P. gingivalis treated cells).

Article Snippet: For intracellular staining, a cytofix/cytoperm kit was used (Becton Dickinson) with fluorescently labeled antibodies against transcription factors: Alexa Fluor®488 Mouse Anti-Stat3 (pY705; BD Biosciences) and PE-IRF4 Monoclonal Antibody (3E4)(eBioscience).

Techniques: Activation Assay, Activity Assay, Staining, Flow Cytometry, Standard Deviation

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: IL-6 Signaling Landscape in Primary Human T Cells (A and B) STAT1 and STAT3 phosphorylation in response to various doses (A) and exposure time (B) of IL-6 stimulation in resting and activated primary human CD4 + and CD8 + T cells. Error bars show mean ± SEM from three individual biological replicas. (C and D) Phospho-FLOW analysis of IL-6 signaling pathways in resting (C) and activated primary human CD4 + and CD8 + T cells treated with HyIL-6 or anti-CD3/CD28 (TCR) + IL-2. ns, cells without any stimulation. Heatmaps show fold change in the level of phosphorylation or protein expression of the different proteins. See also and . (E and F) Effect of JAK inhibition (2 μM tofacitinib) on the phosphorylation of STAT1 (E) and STAT3 (F) Tyr701 and Ser727 in resting and activated primary human CD4 + and CD8 + T cells. Error bars show mean ± SEM from three individual biological replicas.

Article Snippet: Mouse anti-STAT3-APC (Clone M59-50) , BD Biosciences , Cat#560392; AB_1645463.

Techniques: Expressing, Inhibition

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: STAT1 and STAT3 HyIL-6-Induced Ser727 Phosphorylation Is CDK8/9 Mediated (A and B) Spider plots showing pTyr701 STAT1 (A) or pTyr705 STAT3 (B) (blue line) and pSer727 STAT1 (A) or pSer727 STAT3 (B) (red line) MFI normalized to HyIL-6-treated cells in the presence of different inhibitors in human primary CD4 + Th-1 cells. (C) Effect of different mTOR inhibitors on the STAT1 (top panel) and STAT3 (bottom panel) Ser727 phosphorylation induced by HyIL-6 in human primary CD4 + T cells. (D) Effect of ATM inhibitor (KU53933) and DNA-PK inhibitor (KU57788) on the STAT1 (top panel) and STAT3 (bottom panel) Ser727 phosphorylation induced by HyIL-6 in human primary CD4 + T cells. (E) Effect of different CDK inhibitors on the STAT3 Tyr705 (top panel) and STAT3 Ser727 (bottom panel) phosphorylation induced by HyIL-6 in human primary CD4 + T cells. For all experiments, quantitative data were calculated from three individual biological replicates. Error bars show mean ± SEM.

Article Snippet: Mouse anti-STAT3-APC (Clone M59-50) , BD Biosciences , Cat#560392; AB_1645463.

Techniques:

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: PLA Analysis of the Interaction of STAT3 and CDK8/9 Induced upon HyIL-6 Stimulation in Human Primary CD4 + Th-1 Cells (A and B) Kinetics of the STAT3/CDK8 (A) or STAT3/CDK9 (B) interaction induced by 20 nM HyIL-6 in human primary CD4 + Th-1 cells. Scale bars, 20 μm. Statistical significance was calculated by one-way ANOVA. (C and D) STAT3/CDK8 (C) or STAT3/CDK9 (D) interactions were analyzed by PLA upon 20 nM HyIL-6 stimulation in the absence or presence of 2 μM MSC2530818 or 2 μM flavopiridol or upon treatment with the inhibitor only. Scale bars, 20 μm. Statistical significance was calculated by unpaired t test. White arrows in A to D indicate examples of cells where interaction signal was detected. Cumulative plots from n = 15 pictures alongside show the percentage of positive cells. Error bars show mean ± SEM. The p values were calculated based on non-parametric two-tailed Wilcoxon rank-sum test against the control group (first bar on the left). (E) STAT3/CDK9 interaction analyzed by PLA upon 20 nM HyIL-6 stimulation in STAT3 KnD Hut78 cells reconstituted with STAT3 WT-GFP (top panel) or STAT3 S727A-GFP (bottom panels). White arrows indicate examples of cells expressing the recombinant protein and where the STAT3/CDK9 interaction was detected by PLA. Scale bars, 20 μm. Graphs alongside show the nuclear GFP MFI normalized to unstimulated cells (top graph) or the nuclear STAT3/CDK9 PLA MFI in GFP-positive cells normalized to unstimulated cells (bottom graph). Quantitative data generated from n = 15 pictures. Error bars show mean ± SEM.

Article Snippet: Mouse anti-STAT3-APC (Clone M59-50) , BD Biosciences , Cat#560392; AB_1645463.

Techniques: Two Tailed Test, Expressing, Recombinant, Generated

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Transcriptional Program Elicited by Interplay between HyIL-6 and CDK8 in Human Primary CD4 + Th-1 Cells (A) Number of differentially expressed genes (DEGs; fold chang,e >1.5; p < 0.05) between unstimulated versus HyIL-6-, mesenchymal stem cell (MSC)-, or HyIL-6+MSC-stimulated Th-1 cells in three biological replicates. (B) Scatterplot showing mean gene expression values (n = 3) before (x axis) and after indicated stimulation (y axis). Upregulated (red) and downregulated (blue) genes are highlighted. (C) Representative gene expression across different stimulation. Bars show mean ± SEM. (D) Gene set enrichment analysis (GSEA) ( Subramanian et al., 2005 ) plots for STAT3 upregulated genes (GEO: GSE21670) comparing stimulated versus unstimulated Th-1 transcriptomes. NES, normalized enrichment score; FDR, false discovery rate. (E) Violin plot showing the mean STAT3 binding intensity in n = 2,585 STAT3-bound regions across different stimulations. Peaks are identified by comparing HyIL-6+MSC stimulation and input. The p values were determined by two-tailed Wilcoxon rank-sum test ( ∗∗∗∗ p < 0.0001). (F) Representative loci showing STAT3 binding across different stimulations. The height of the tracks are indicated at bottom-right corner of the plots. (G) GSEA plots for 475 STAT3-bound genes comparing stimulated versus unstimulated Th-1 transcriptomes.

Article Snippet: Mouse anti-STAT3-APC (Clone M59-50) , BD Biosciences , Cat#560392; AB_1645463.

Techniques: Expressing, Binding Assay, Two Tailed Test

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet: Role of CDK8 Ser727 Phosphorylation of STAT3 in Th-17 Differentiation In Vitro (A) Experimental workflow for human Th-17 differentiation in vitro from isolated human resting CD4 + T cells. (B and C) Dot plot representations of IL-17- and IFNγ-positive cells in populations grown in the presence of HyIL-6 (B) or HyIL-6 + MSC2530818 (C). (D) IL-17-positive cells were identified by flow cytometry in untreated cells or cells treated with 2 μM MSC2530818. Data are percentage of positive cells ± SEM in four biological replicates; p values were calculated using a paired t test. (E) As in (D) but for IFNγ-positive cells. (F) Amount of IL-17 ± SEM in four biological replicates detected in growth media following growth of cells minus or plus inhibitor. (G) Amount of IFNγ ± SEM in four biological replicates detected in growth media following growth of cells minus or plus inhibitor. Statistical significance was calculated by unpaired t test.

Article Snippet: Mouse anti-STAT3-APC (Clone M59-50) , BD Biosciences , Cat#560392; AB_1645463.

Techniques: In Vitro, Isolation, Flow Cytometry

Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet:

Article Snippet: Mouse anti-STAT3-APC (Clone M59-50) , BD Biosciences , Cat#560392; AB_1645463.

Techniques: Purification, Recombinant, Software