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: Journal of biomolecular screening

Article Title: Comparison of 2 cell-based phosphoprotein assays to support screening and development of an ALK inhibitor.

doi: 10.1177/1087057110394657

Figure Lengend Snippet: FIG. 5. Dual-parameter flow cytometric analysis of the response to an ALK inhibitor. ALK-positive Karpas-299 and SupM2 cells and ALK- negative HT cells were stained concurrently with 2 analytes and treated with DMSO or 0.1 µM, or 1 µM of PF-2341066. IC50 values were cal- culated from change in median fluorescence intensity (MFI) for each analyte. (A) Background levels of total ALK, pY1604 ALK, pY705 STAT3, and pY473 AKT staining in DMSO-treated cells. (B) Karpas-299 and SupM2 cells stained concurrently with pY1604 ALK and pY705 STAT3 antibod- ies demonstrate inhibition of both analytes when treated with PF-2341066. (C) Karpas-299 and SupM2 cells stained concurrently with pY473 AKT and total ALK antibodies do not show inhibition of pY473 AKT when treated with PF-2341066.

Article Snippet: Phosflow cell-based pY1604 ALK assay cells (250,000 per well) were plated in 100 μl of culture medium in 96-well plates. each well contained 1 μl of test compound at 100× concentration in 100% DMso, for a final reaction concentration of 1% DMso. cells and compound were incubated at 37°c for 1 h, fixed with 4% paraformaldehyde for 30 min at room temperature, and then permeabilized overnight with ice-cold 90% Meoh at –20°c. primary antibodies py1604 alK rabbit polyclonal antibody, py473 aKt (D9e) rabbit monoclonal, and py705 stat3 (3e2) mouse monoclonal antibody from cell signaling technologies (Danvers, Ma) and total alK at Kungl Tekniska Hogskolan / Royal Institute of Technology on March 10, 2015jbx.sagepub.comDownloaded from 166 www.slas.org Journal of Biomolecular Screening 16(2); 2011 (5a4) mouse monoclonal antibody from abcam (cambridge, Ma) were added for 1 h at room temperature. fluorescein isothiocyanate- or phycoerythrin-conjugated secondary antibodies (invitrogen) were added for 30 min at room temperature.

Techniques: Staining, Fluorescence, Inhibition

Journal: BMC Cancer

Article Title: RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

doi: 10.1186/1471-2407-13-463

Figure Lengend Snippet: Oxaliplatin (OXP) reduces the phosphorylation of STAT3 and RKIP that is induced by interleukin-6 (IL-6). Western blot analysis of: (A) the indicated proteins in HCT116 cells treated with IL-6 (40 ng/ml) for 1–6 h. (B) HCT116 cells treated with 300 μM OXP, IL-6 (40 ng/ml) in serum free medium or the combination for 16 h and analyzed for the indicated proteins via Western blot analysis. (C) Cell extracts were prepared after 18 h following treatment with OXP, IL-6 or the combination for flow cytometric analysis to examine binding to 7-AAD and annexin-V (% Apoptosis). The % Apoptosis of each sample is indicated in the top right corner of every panel: a) untreated control cells (0.20%); b) 5 μM STS (70.94%); 300 μM OXP (31.04%); d) IL-6 (4.01%); e) OXP + IL-6 (19.56%). STS is the positive control. The figure is representative of part of 1 experiment performed in duplicate. The experiment was repeated twice.

Article Snippet: The antibodies to STAT3 (sc-482), pRKIP (sc-32623), gp130 (SC-655) and actin (SC-1616) were purchased from Santa Cruz Biotechnology; STAT3 pY705 (9131S) and PARP (9542S) from Cell Signaling Technology; RKIP (07–137) and Histone 2AX (07–67) from Millipore, Milford, MA.

Techniques: Phospho-proteomics, Western Blot, Binding Assay, Control, Positive Control

Journal: BMC Cancer

Article Title: RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

doi: 10.1186/1471-2407-13-463

Figure Lengend Snippet: Camptothecin (CPT) reduces phosphorylation of STAT3 and RKIP induced by interleukin-6 (IL-6). (A) Western blot analysis of: a dose-dependent reduction of STAT3 phosphorylation after IL-6 treatment then treated with 250–750 nM CPT; HCT116 cells treated with 250 nM CPT and 40 ng/ml IL-6 for 12 h. (B) The same co-treatment experiment as in (A) was repeated with HCT116 cells treated with 250 nM CPT and 40 ng/ml IL-6 for 12 h. Western blot analysis was performed to examine the protein levels of pRKIP, RKIP and actin. (C) Cell extracts were prepared after 18 h following treatment with CPT, IL-6 or the combination for flow cytometric analysis to examine binding to 7-AAD and annexin-V (% Apoptosis). The % Apoptosis of each sample is indicated in the top right corner of every panel: a) untreated control cells (0.48%); b) CPT (17.68%); c) IL-6 (0.64%); d) CPT + IL-6 (10.94%). The figure is representative of part of 1 experiment performed in duplicate. The experiment was repeated twice. (D) HCT116 cells were transfected with an IRF-1 reporter plasmid for STAT3 activation. After 48 h, the cells were washed and treated with 40 ng/ml IL-6, 250 nM CPT, or the combination. After 24 h, samples were harvested and washed twice before being lysed and combined with a luciferase assay reporter. The data is reported as the mean +/− s.d. of 2 independent experiments performed in triplicate. A paired t-test was performed to analyze the increase in STAT3 transcription of IL- 6 treated experimental samples when compared to vehicle (CTR): *IL-6, p < 0.000012; or decrease when comparing IL-6 to samples treated with **IL-6 and CPT, p < 0.0002.

Article Snippet: The antibodies to STAT3 (sc-482), pRKIP (sc-32623), gp130 (SC-655) and actin (SC-1616) were purchased from Santa Cruz Biotechnology; STAT3 pY705 (9131S) and PARP (9542S) from Cell Signaling Technology; RKIP (07–137) and Histone 2AX (07–67) from Millipore, Milford, MA.

Techniques: Phospho-proteomics, Western Blot, Binding Assay, Control, Transfection, Plasmid Preparation, Activation Assay, Luciferase

Journal: BMC Cancer

Article Title: RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

doi: 10.1186/1471-2407-13-463

Figure Lengend Snippet: Camptothecin blocks STAT3 activation and the interaction of STAT3 and the gp130 receptor. (A) Western blot analysis for the indicated proteins from HCT116 cells transfected with STAT3 cDNA and then treated with 250 nM CPT for 16 h. (B) HCT116 cells were transfected with an IRF-1 reporter plasmid to measure STAT3 activation along with JAK1 and 2 cDNAs. After 48 h, cells were washed and treated 250 nM CPT. After 24 h, the samples were harvested and washed twice before being lysed and combined with a luciferase assay reporter. The data is reported as the mean +/− s.d. of 2 independent experiments performed in triplicate. (C) HCT cells were transfected with STAT3 cDNA and gp130 cDNA. After 48 h, Samples were treated with 40 ng/ml IL-6 or IL-6 and 250 nM CPT. Samples were divided and either saved for Western blot analysis (input) or incubated with an antibody to gp130 for 6 h. Protein G agarose beads were added and the samples rotated over night. Western blot analysis was performed using the IP supernatant and examined for the indicated proteins. In comparison to empty vector controls (EV), the relative activity of STAT3 transcription was increased by: *JAK1, p < 0.00005; **JAK2, p < 0.0001. In the presence of CPT, JAK1-mediated STAT3 transcription was inhibited #JAK1 + CPT p < 0.0002 and JAK2 inhibited ## JAK2 + CPT p < 0.0003. The data represents the mean +/− s.d. of 2 independent experiments performed in duplicate.

Article Snippet: The antibodies to STAT3 (sc-482), pRKIP (sc-32623), gp130 (SC-655) and actin (SC-1616) were purchased from Santa Cruz Biotechnology; STAT3 pY705 (9131S) and PARP (9542S) from Cell Signaling Technology; RKIP (07–137) and Histone 2AX (07–67) from Millipore, Milford, MA.

Techniques: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase, Incubation, Comparison, Activity Assay

Journal: BMC Cancer

Article Title: RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

doi: 10.1186/1471-2407-13-463

Figure Lengend Snippet: Contingency analysis of cytoplasmic phosphorylated RKIP (c-pRKIP), nuclear phosphorylated RKIP (n-pRKIP) and nuclear STAT3 (n-STAT3) expression and tumor Grade and LVI status

Article Snippet: The antibodies to STAT3 (sc-482), pRKIP (sc-32623), gp130 (SC-655) and actin (SC-1616) were purchased from Santa Cruz Biotechnology; STAT3 pY705 (9131S) and PARP (9542S) from Cell Signaling Technology; RKIP (07–137) and Histone 2AX (07–67) from Millipore, Milford, MA.

Techniques: Expressing

Journal: BMC Cancer

Article Title: RKIP phosphorylation and STAT3 activation is inhibited by oxaliplatin and camptothecin and are associated with poor prognosis in stage II colon cancer patients

doi: 10.1186/1471-2407-13-463

Figure Lengend Snippet: Nuclear STAT3 in stage II colon cancer is associated with negative pathological features. (A) Representative examples of immunohistochemical staining for STAT3 showing strongly positive (3+) and negative levels. Bar is 100 micron. (B) Nuclear STAT3 is associated with higher grade tumors, where fewer than 5% of high grade tumors received a negative staining score compared to nearly 22% of low grade tumors (p < 0.0266). (C) Nuclear STAT3 is associated with lymphovascular invasion. No patients with LVI had negative nuclear STAT3 staining (p < 0.0218). (D) High levels of cytoplasmic STAT3 are associated with tumor grade, with higher levels correlating with higher grade (p = 0.03).

Article Snippet: The antibodies to STAT3 (sc-482), pRKIP (sc-32623), gp130 (SC-655) and actin (SC-1616) were purchased from Santa Cruz Biotechnology; STAT3 pY705 (9131S) and PARP (9542S) from Cell Signaling Technology; RKIP (07–137) and Histone 2AX (07–67) from Millipore, Milford, MA.

Techniques: Immunohistochemical staining, Staining, Negative Staining

Journal: Cell metabolism

Article Title: STAT3 Activation-Induced Fatty Acid Oxidation in CD8 + T Effector Cells Is Critical for Obesity-Promoted Breast Tumor Growth

doi: 10.1016/j.cmet.2019.10.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-pY705-STAT3 , Cell Signaling Technology , Cat#9145S; RRID: AB_2491009.

Techniques: Purification, Control, Virus, Recombinant, Staining, Cell Isolation, cDNA Synthesis, SYBR Green Assay, Proliferation Assay, Mouse Assay, Software, Microscopy

Journal: The Journal of investigative dermatology

Article Title: B7-H3 Associated with Tumor Progression and Epigenetic Regulatory Activity in Cutaneous Melanoma

doi: 10.1038/jid.2013.114

Figure Lengend Snippet: A : Binding site of miR-29c(bottom strand) to B7-H3 mRNA 3′UTR position from 1338 to 1346(top strand) (9 bp in length). B : B7-H3 IHC staining score vs. miR-29c mRNA expression in melanoma TMA samples (paired T-test analysis). C : Flow cytometry analysis of B7-H3 expression in a melanoma cell line with transfection of miR-29c mimic and with a negative miRNA control. D : Quantitation of B7-H3 expression change after transfection with miR-29c. E : Relationship of IHC staining score of p-STAT3 versus B7-H3 in metastatic melanoma PEAT. F : Western blot of STAT3 and p-STAT3 in a melanoma cell line transfected with miR-29c mimic and with negative miRNA control. G . Western blot of STAT3, p-STAT3 and cyclin D1 in a melanoma cell lines transfected with B7-H3 shRNA or with shRNA control.

Article Snippet: Tissue sections were then blocked with goat serum(10%) for 1hr followed by incubation with rabbit anti-pY705-STAT3 monoclonal Ab(Cell Signaling, Danvers, MA) overnight at 4°C.

Techniques: Binding Assay, Immunohistochemistry, Expressing, Flow Cytometry, Transfection, Quantitation Assay, Western Blot, shRNA