Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: PARP-1 influences STAT6 protein level and regulates its occupancy on the gata-3 promoter following IL-4 stimulation. A WT and PARP-1 −/− splenocytes isolated from C57BL/6 J mice were stimulated with IL-4 for the indicated time points. Protein lysate were analyzed for STAT6, pSTAT6 (Y641), PARP-1 and Actin. The bands of STAT6 were quantified and expressed as percent change compared with their respective untreated control/actin (right panel). B IL-4-treated cells and relative controls undergo subcellular fractionation and then protein extracts were analyzed for STAT6, Lamin B and Actin. C WT and PARP-1 −/− splenocytes isolated from C57BL/6 J mice were stimulated with IL-4 for the indicated intervals of time. ChIP for STAT6 was performed and immunoprecipitated DNA was analyzed using qPCR targeting the proximal or distal promoter of gata-3. Data are representative of at least three independent experiments. *, # Significant difference from respective controls or experimental sample, respectively ( p < 0.05)

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Isolation, Fractionation, Immunoprecipitation

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: STAT6 interacts with and is PARylated by PARP-1 in a cell-free system with recombinant proteins and in IL-4 stimulated mouse splenocytes. A Workflow of the cell-free PARylation reaction. B Ni–NTA pull down for hSTAT6 was performed and samples were examined with antibodies against PAR, or STAT6. C IL-4-treated splenocytes and controls were collected at the indicated time points. Protein extracts were subjected to immunoprecipitation (IP) with antibodies to STAT6. Subsequently, an immunoblot analysis of the IP material or input proteins was performed using antibodies against STAT6, p-STAT6 (Y641), PAR, PARP-1, or Actin. Data are representative of at least 3 independent experiments

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Recombinant, Immunoprecipitation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: STAT6 is selectively degraded by Calpain-1 but its PARylation protects it against this process . A Recombinant poly-His-STAT6 was phosphorylated in vitro by active JAK3 or left unphosphorylated prior to incubation with calpain-1 for 10 min. The reaction was stopped with sample buffer and analyzed by immunoblot analysis. The bands were quantified and expressed as percent change compared with their respective untreated control (bottom panel). B Calpain-1 was incubated with STAT6, JAK3, JAK1, PARP-1, or PARG for 40 min. Immunoblot analyses were performed with antibodies against STAT6, PARP-1, JAK1, JAK3, or PARG. C Calpastatin was added to the cell free reaction mix containing STAT6 and Calpain-1. Samples were then subjected to immunoblot analysis with antibodies to the respective proteins. D JAK3-mediated STAT6 phosphorylation, PARylation, and calpain enzymatic reactions were performed in this order. Immediately after the phosphorylation the reaction was divided in 4 parts.Then, immunoblot analysis was carried out with antibodies against STAT6, p-STAT6 (Y641), PARP-1, or PAR. E Recombinant STAT6 was incubated with JAK3 in the presence of calpain-1 with or without NAD + . STAT6 degradation was assessed by immunoblot analysis

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Recombinant, In Vitro, Incubation, Western Blot

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: STAT6 is degraded by AA starvation-induced autophagy . A – B Jurkat, MEF and PM1 cells were exposed to AA starvation media for the indicated time and then processed for immunoblot analysis. Antibodies against STAT6, LC3, actin, or tubulin were used. C Jurkat cells were cultured for 6 h in starvation media and then supplemented with AA for the indicate times. Protein extracts were subjected to immunoblot analysis with antibodies to STAT6, tubulin, or LC3. D The blot from ( A ) was re-probed with antibodies to STAT1, STAT2, STAT3, STAT4 or PARP-1. E CAPNS1 −/− cells expressing human CAPNS1 or empty vector were subjected to AA starvation for the indicated times. Protein extracts were then subjected to immunoblot analysis with antibodies to STAT6, CAPNS1, LC3 or actin. F Putative CMA-targetable motifs on STAT6. G Jurkat cells subjected to AA starvation for 6 h were concomitantly treated with CMA inducers or inhibitors or calpastatin. Levels of STAT6 and autophagy status were assessed by immunoblot analysis of protein extracts with antibodies to STAT6, LC3 or actin

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Western Blot, Cell Culture, Expressing, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: IL-4 treatment protects STAT6 from autophagy-induced degradation and PARP-1 inhibition abrogates such protection without affecting autophagy . A Splenocytes isolated from WT C57BL/6 J mice were stimulated with IL-4 in the absence or presence of olaparib for the indicated time points. Protein lysates were subjected to immunoblot analysis with antibodies to STAT6 or Actin. B Jurkat cells were exposed to AA starvation media then supplemented with different percentages of AAs in the presence or absence of IL-4 or the PARP inhibitor, olaparib, for 12 h. Protein extracts were then subjected to immunoblot analysis with antibodies to STAT6, p62 or Tubulin. The brackets on the left indicate that the two sets of panels were of same samples but run on two different gels. C Jurkat were cultured in starvation media then supplemented with 25% AAs and treated for 12 h with IL-4, olaparib, calpastatin or combinations of the different agents. Not starved cells were used as control. Protein extracts were then subjected to immunoblot analyses with antibodies to STAT6, STAT4, p62 or actin. D The intensity of the STAT6 bands showed on ( B ) was quantified using ImageJ-Fiji and results were normalized to respective Actin band intensity

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Inhibition, Isolation, Western Blot, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Promotion of a synthetic degradation of activated STAT6 by PARP-1 inhibition: roles of poly(ADP-ribosyl)ation, calpains and autophagy

doi: 10.1186/s12967-022-03715-x

Figure Lengend Snippet: Synthetic degradation of IL-4-activated STAT6 upon PARP inhibition and its association with calpains and autophagy. IL-4 binds to its receptor (upon the dimerization of the IL-R4α and γC subunits) leading to the recruitment and subsequent activation of JAK1/3 kinases, which culminates in the phosphorylation of STAT6. Phosphorylated STAT6 monomers then dimerize and translocate to the nucleus where they occupy the gata-3 gene promoter. During the early stages after IL-4 stimulation, PARP-1 expression is required for a persistent occupancy of the gata-3 promoter by the STAT6 dimer. Whether PARylation is required here is not clear. At a later stage, phosphorylated STAT6 is PARylated, which protects it from calpain-mediated degradation. Inhibition of PARylation by PARP inhibitors renders STAT6 susceptible to degradation. Autophagy also promotes degradation of STAT6 and is protected by IL-4 stimulation and subsequent PARylation. CMA appears to be the major mechanism by which STAT6 is degraded during autophagy. The increased susceptibility of phosphorylated STAT6 for degradation by calpains upon PARP inhibition may be considered as artificial or synthetic, hence our proposal of naming this process “ synthetic protein degradation ”

Article Snippet: Recombinant human STAT6 (400 ng) (Sino Biological) was incubated with 100 ng of recombinant human JAK3 (Active Motif) in kinase buffer and then incubated with 1.5 mM ATP (Affymetrix) to start the reaction for 30 min at 37 °C.

Techniques: Inhibition, Activation Assay, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma

doi: 10.1111/jcmm.12014

Figure Lengend Snippet: Representative micrographs stained with haematoxylin & eosin to show ( A ) sheets of differentiated columnar epithelium obtained by Rhinoprobe™ curettage sampling (40×), and ( B ) the presence of beating cilia (indicated by arrow) which were readily discernible upon placement in culture (100×). RT-PCR analysis of NEC's was carried out at the time of sampling to compare ( C ) STAT6, ( D ) CCL26 and ( E ) MUC5AC mRNA expression between non-asthmatic (closed bars) and asthmatic (open bars) donor groups. Values presented are the mean fold-change, relative to the mean δCt value for the non-asthmatic group ± SD (non-asthmatic control: n = 4, asthmatic donors: n = 19). Data were analysed by Mann–Whitney U -test and values differ from non-asthmatic: * P < 0.05.

Article Snippet: Total RNA was isolated using Tri-Reagent® (Sigma-Aldrich) and real-time RT-PCR performed with TaqMan gene expression assays: STAT6 Hs00598625_m1; CCL26 Hs00171146_m1; CHI3L1 Hs00609691_m1; MUC5AC Hs00873651_mH; PROM1 Hs01009257_m1; GAPDH Hs99999905_m1; β-actin Hs99999903_m1 (Life Technologies).

Techniques: Staining, Sampling, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma

doi: 10.1111/jcmm.12014

Figure Lengend Snippet: ( A ) Primary nasal epithelial cells from non-asthmatic (closed bars) or asthmatic (open bars) donors were cultured in epithelial growth medium for 30 days. Cells were harvested for RNA extraction to measure the relative expression of STAT6, CHI3L1, MUC5AC and PROM1 compared with expression at the time of nasal sampling (day 0). ( B ) Primary nasal epithelial cells from asthmatic donors were cultured in epithelial growth medium for 72 hrs. Cells were harvested for RNA extraction at 24, 48 or 72 hrs to measure the relative expression of STAT6 ( ) and CCL26 ( ) compared with expression at the time of nasal sampling (day 0). Values presented are the mean fold -change, relative to the mean δCt value for each group at day 0 ± SD. (A) Non-asthmatic: n = 4, asthmatic: n = 5. (B) Asthmatic: n = 3. Data were analysed by Student's t -(STAT6) or Mann–Whitney U -test (CHI3L1, MUC5AC, PROM1 and CCL26) and values differ from time of sampling (day 0): * P < 0.05.

Article Snippet: Total RNA was isolated using Tri-Reagent® (Sigma-Aldrich) and real-time RT-PCR performed with TaqMan gene expression assays: STAT6 Hs00598625_m1; CCL26 Hs00171146_m1; CHI3L1 Hs00609691_m1; MUC5AC Hs00873651_mH; PROM1 Hs01009257_m1; GAPDH Hs99999905_m1; β-actin Hs99999903_m1 (Life Technologies).

Techniques: Cell Culture, RNA Extraction, Expressing, Sampling, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma

doi: 10.1111/jcmm.12014

Figure Lengend Snippet: Primary nasal epithelial cells from non-asthmatic (closed bars) or asthmatic (open bars) donors were transfected with STAT6-targeting siRNA (si372), scrambled control siRNA (SC) or transfection reagent only (TF) either in the presence or absence of IL-13 (50 ng/ml). Cells were harvested for RNA extraction to measure the percentage inhibition of STAT6 ( A ) and relative expression of CCL26 ( B ) following 72-hr treatment. Values presented are percentage inhibition (STAT6) or mean fold change (CCL26), relative to the TF group ± SD (non-asthmatic controls: n = 4, asthmatic donors: n = 5). Data were analysed by Student's t -(STAT6) or Mann–Whitney U -test (CCL26) and values differ from TF: * P < 0.05.

Article Snippet: Total RNA was isolated using Tri-Reagent® (Sigma-Aldrich) and real-time RT-PCR performed with TaqMan gene expression assays: STAT6 Hs00598625_m1; CCL26 Hs00171146_m1; CHI3L1 Hs00609691_m1; MUC5AC Hs00873651_mH; PROM1 Hs01009257_m1; GAPDH Hs99999905_m1; β-actin Hs99999903_m1 (Life Technologies).

Techniques: Transfection, Control, RNA Extraction, Inhibition, Expressing, MANN-WHITNEY

Journal: Journal of Cellular and Molecular Medicine

Article Title: Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma

doi: 10.1111/jcmm.12014

Figure Lengend Snippet: RPMI 2650 nasal epithelial cells were cultured as monolayers at an air–liquid interface in epithelial growth medium for 20 days, followed by further culture in epithelial growth medium containing 5 ng/ml IL-13 for 5 days. Cells were harvested for RNA extraction at days 1, 3, 6, 8, 10, 13, 15, 17 and daily from 20 to 25 to measure the relative mRNA expression of ( A ) CHI3L1, ( B ) MUC5AC, ( C ) PROM1, ( D ) STAT6 and ( E and F ) CCL26 by RT-PCR. ( G ) RPMI 2650 nasal epithelial cells grown under submerged (closed bars) or ALI (open bars) culture conditions were transfected with STAT6-targeting siRNA (si372), scrambled control siRNA (SC) or transfection reagent only (TF). Cells were harvested for RNA extraction to measure the percentage inhibition of STAT6 following 72-hr treatment. Values presented are mean fold change (A–F), relative to day 0, or percentage inhibition of STAT6 mRNA (G), relative to TF, ±SD ( n = 3). Data were analysed by Student's t - (percentage inhibition) or Mann–Whitney U -test (fold -change) and values differ from day 0 or TF: * P < 0.05.

Article Snippet: Total RNA was isolated using Tri-Reagent® (Sigma-Aldrich) and real-time RT-PCR performed with TaqMan gene expression assays: STAT6 Hs00598625_m1; CCL26 Hs00171146_m1; CHI3L1 Hs00609691_m1; MUC5AC Hs00873651_mH; PROM1 Hs01009257_m1; GAPDH Hs99999905_m1; β-actin Hs99999903_m1 (Life Technologies).

Techniques: Cell Culture, RNA Extraction, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Inhibition, MANN-WHITNEY

Journal: Respiratory Research

Article Title: Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

doi: 10.1186/s12931-014-0164-4

Figure Lengend Snippet: OSM and other gp130 cytokine stimulation and HASMC responses. (A-C) HASMC cultures were prepared and stimulated as in Figure with the indicated cytokines and concentrations of 10 ng/ml for each. Data shown are from one of two individual cell lines that showed identical trends. 24-hour culture supernatants were assessed by ELISA for levels of MCP-1/CCL-2 (A), IL-6 (B) and eotaxin-1 (C) . *** p < 0.001 indicates statistical significance comparing indicated cytokine combinations to either cytokine alone using One-Way ANOVA with Bonferroni’s post-test. (D) HASMC cultures were stimulated with OSM, LIF, IL-11, IL-31 or IL-6 (10 ng/ml) for 20 minutes and cell lysates were prepared for immunoblots as described in methods. The lysates were probed for p-Y-STAT1, STAT1, p-Y-STAT3, STAT3, p-Y- STAT5, STAT5, p-Y-STAT6, p-Ser-Akt, p-T/Y-p38, p-T/Y-JNK and Actin as indicated. (E) Quantitative analysis of band intensity using densitometry (ImageJ) corrected to Actin or total protein for each probe and expressed as fold change relative to control (unstimulated).

Article Snippet: Primary antibodies specific for Actin and STAT6 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Respiratory Research

Article Title: Regulation of IL-17A responses in human airway smooth muscle cells by Oncostatin M

doi: 10.1186/s12931-014-0164-4

Figure Lengend Snippet: STAT3, p38 (MAPK) and Akt activation in HASMC and effects of inhibition on HASMC responses. (A) HASMC cultures were prepared as previously outlined for immunoblots of lysates of cells stimulated for 20 minutes with 5 ng/ml of IL-4, IL-13 and IL-17A (and/or 1 ng/ml of OSM). Total cell lysates from 3 different cell lines were probed for p-Y-STAT3, STAT3, p-Y-STAT6, STAT6, p-S-Akt, Akt, p-T/Y-p38, and total p38 as indicated (one representative cell line shown). (B) Quantification of band intensity using densitometry (ImageJ) corrected to total protein for each probe and expressed as the average (3 cell lines) fold change relative to control (un-stimulated) * p <0.05 using one-tail t- test. (C) . HASMC cultures were prepared as previous for immunoblots of lysates of cells stimulated for 20 minutes with 1 ng/ml OSM, with or without pre-incubation with 1.25, or 2.5 or 5 uM Stattic or its vehicle DMSO (Veh). The lysates were probed for p-Y-STAT1, total STAT1, p-Y-STAT3, p-Y-STAT5, p-T/Y-p38, total p38, p-S-Akt and actin as indicated.

Article Snippet: Primary antibodies specific for Actin and STAT6 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Inhibition, Western Blot, Control, Incubation

Journal: Cell Death & Disease

Article Title: M2 macrophage-mediated interleukin-4 signalling induces myofibroblast phenotype during the progression of benign prostatic hyperplasia

doi: 10.1038/s41419-018-0744-1

Figure Lengend Snippet: a SMAD3, STAT6, AKT and ERK phosphorylation levels in PrPF-early, PrPF-control and PrPF-old cells treated with the conditioned media (CMs) obtained from the THP-1-derived M2 macrophage–fibroblast co-cultures. b SMAD3, STAT6, AKT and ERK phosphorylation levels in PrPF-early, PrPF-control and PrPF-old cells treated with 1 ng/ml TGFβ1. c SMAD3 phosphorylation levels in PrPF-early cells following the treatment with CM pretreated for 3 h with 1 μg/ml anti-TGFβ1 antibody. Additionally, PrPF-early cells were pretreated with 1 μM SD208, a small-molecule ALK5 inhibitor of the TGFβ1R1/SMAD2/3 interaction, for 3 h. IgG isotype antibody (1 μg/ml) was used as a control. d JAK/STAT6, PI3K/AKT, SMAD3 and MAPK/ERK signalling following the treatment with 10 ng/ml IL4 in PrPF-early cells. e STAT6, AKT and ERK phosphorylation following the antibody-induced inhibition of IL4 for 3 h, prior to the treatment of PrPF-early cells with these media. CMs pretreated with 1 μg/ml IgG isotype antibody were used as controls. f Schematic illustration of the mechanisms underlying M2 macrophage-induced development of myofibroblast phenotype through the activation of TGFβ1 and IL4 signalling

Article Snippet: The primary antibodies against α-SMA (ab7817, 1:300 dilution), collagen I (ab138492, 1:1000 dilution), GAPDH (ab8245, 1:5000 dilution), Smad3 (ab40854, 1:1000 dilution) and phospho-Smad3 (Ser 423 and Ser 425, ab52903, 1:1000 dilution) were purchased from Abcam (MA, USA); STAT6 (#9362, 1:1000 dilution), phospho-STAT6 (Tyr641, #9361, 1:1000 dilution), Akt (#9272, 1:1000 dilution), phospho-Akt (Ser473, #9271, 1:1000 dilution), ERK 1/2 (p44/42 MAPK, #4695, 1:1000 dilution) and phospho-Erk1/2 (Thr202/Tyr204, #4370, 1:1000 dilution) from Cell Signaling Technology (MA, USA) and IL4Rα (YT2337, 1:1000 dilution), TGFβ RI (YT4627, 1:1000 dilution) and TGFβ RII (YT4628, 1:1000 dilution) from ImmunoWay (TX, USA).

Techniques: Phospho-proteomics, Control, Derivative Assay, Inhibition, Activation Assay

Journal: Oncogene

Article Title: Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer.

doi: 10.1038/sj.onc.1210222

Figure Lengend Snippet: Figure 1 Inhibition of STAT6 expression by siRNA decreases constitutive COX-2 expression. Cells were transfected with STAT6 or negative control siRNA. Cells were lysed 48 h later and cell lysates were subjected to Western blot.

Article Snippet: The antibodies used in this study includes COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), STAT6 for Western blot (Cell Signaling Technology, Danvers, MA, USA), for immunofluorescence (IF) (BD Pharmingen, San Diego, CA, USA), for ChIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT6 (Cell Signaling Technology), p300 for Western blot, (Upstate, Lake Placid, NY, USA), for ChIP (Santa Cruz Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology), mouse immunoglobulin (Ig)G1, k Ig (BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (Advanced ImmunoChemical, Long Beach, CA, USA), Cy3conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and PARP (Cell Signaling Technology).

Techniques: Inhibition, Expressing, Transfection, Negative Control, Western Blot

Journal: Oncogene

Article Title: Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer.

doi: 10.1038/sj.onc.1210222

Figure Lengend Snippet: Figure 2 Constitutive COX-2 expression is decreased in STAT6 DN cell lines and is increased in STAT6Y641W cell lines. Cells from STAT6 DN (a) or STAT6Y641W (b) stable cell lines, corres- ponding vector controls (V) or parental cells (P) were cultured under normal culture condition and harvested. Cell lysates were subjected to Western blot.

Article Snippet: The antibodies used in this study includes COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), STAT6 for Western blot (Cell Signaling Technology, Danvers, MA, USA), for immunofluorescence (IF) (BD Pharmingen, San Diego, CA, USA), for ChIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT6 (Cell Signaling Technology), p300 for Western blot, (Upstate, Lake Placid, NY, USA), for ChIP (Santa Cruz Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology), mouse immunoglobulin (Ig)G1, k Ig (BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (Advanced ImmunoChemical, Long Beach, CA, USA), Cy3conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and PARP (Cell Signaling Technology).

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Cell Culture, Western Blot

Journal: Oncogene

Article Title: Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer.

doi: 10.1038/sj.onc.1210222

Figure Lengend Snippet: Figure 4 Immunofluorescent nuclear staining of STAT6 in H2122 cells. Cells were stained with STAT6 monoclonal antibody or mouse IgG1 isotype control antibody or without the primary antibody. Bar ¼ 8 mm.

Article Snippet: The antibodies used in this study includes COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), STAT6 for Western blot (Cell Signaling Technology, Danvers, MA, USA), for immunofluorescence (IF) (BD Pharmingen, San Diego, CA, USA), for ChIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT6 (Cell Signaling Technology), p300 for Western blot, (Upstate, Lake Placid, NY, USA), for ChIP (Santa Cruz Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology), mouse immunoglobulin (Ig)G1, k Ig (BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (Advanced ImmunoChemical, Long Beach, CA, USA), Cy3conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and PARP (Cell Signaling Technology).

Techniques: Staining, Control

Journal: Oncogene

Article Title: Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer.

doi: 10.1038/sj.onc.1210222

Figure Lengend Snippet: Figure 5 STAT6 and p300 interact and bind to the COX-2 promoter. (a) ChIP assays of STAT6 and p300. The ‘ þ ’ and ‘’ indicates the presence of antibodies for STAT6 or p300 ( þ ) or their IgG control antibodies (–). Each sample DNA was amplified by PCR with primers to the STAT6 binding site or negative control primers to ensure equal amounts of DNA were utilized in the PCR reaction. (b) Co-immunoprecipitation assay of STAT6Y641W and p300. 293 cells were transfected with STAT6Y641W and/or p300. Cell lysates were directly subjected to Western blot to assess the expression of STAT6 and p300. Cell lysates were also immuno- precipitated with p300 antibody and then subjected to Western blotting. The membrane was probed with STAT6 antibody.

Article Snippet: The antibodies used in this study includes COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), STAT6 for Western blot (Cell Signaling Technology, Danvers, MA, USA), for immunofluorescence (IF) (BD Pharmingen, San Diego, CA, USA), for ChIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT6 (Cell Signaling Technology), p300 for Western blot, (Upstate, Lake Placid, NY, USA), for ChIP (Santa Cruz Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology), mouse immunoglobulin (Ig)G1, k Ig (BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (Advanced ImmunoChemical, Long Beach, CA, USA), Cy3conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and PARP (Cell Signaling Technology).

Techniques: Control, Binding Assay, Negative Control, Co-Immunoprecipitation Assay, Transfection, Western Blot, Expressing, Membrane

Journal: Oncogene

Article Title: Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer.

doi: 10.1038/sj.onc.1210222

Figure Lengend Snippet: Figure 6 Unphosphorylated STAT6 increases COX-2 promoter activities and binds to the COX-2 promoter sequence in vitro. (a) Luciferase reporter assay of COX-2 promoter. COX-2 promoter constructs with the presence (1432/ þ 59 bp) and absence (327/ þ 59 bp) of the STAT6 binding site were co-transfected with STAT6Y641W construct or vector control and a renilla luciferase construct into 293 cells. Luciferase activity was presented as the ratio of firefly to renilla luciferase. (b and c) EMSA assay. The COX-2 promoter sequence containing the STAT6 binding site (30 bp) was used as a probe. (b) Nuclear extracts from H2122 cells were incubated with either the biotin-labeled probes alone (far left lane), or together with different molar excess (20-, 50-, 100-fold, respectively) of non-biotin labeled same probes. (c) Nuclear extracts were incubated with the biotin-labeled probes alone (middle lane) or 10-fold molar excess of non-biotin labeled wild-type probes or mutant probes.

Article Snippet: The antibodies used in this study includes COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), STAT6 for Western blot (Cell Signaling Technology, Danvers, MA, USA), for immunofluorescence (IF) (BD Pharmingen, San Diego, CA, USA), for ChIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT6 (Cell Signaling Technology), p300 for Western blot, (Upstate, Lake Placid, NY, USA), for ChIP (Santa Cruz Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology), mouse immunoglobulin (Ig)G1, k Ig (BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (Advanced ImmunoChemical, Long Beach, CA, USA), Cy3conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and PARP (Cell Signaling Technology).

Techniques: Sequencing, In Vitro, Luciferase, Reporter Assay, Construct, Binding Assay, Transfection, Plasmid Preparation, Control, Activity Assay, Incubation, Labeling, Mutagenesis

Journal: Oncogene

Article Title: Unphosphorylated STAT6 contributes to constitutive cyclooxygenase-2 expression in human non-small cell lung cancer.

doi: 10.1038/sj.onc.1210222

Figure Lengend Snippet: Figure 7 Unphosphorylated STAT6 affects cell sensitivity to apoptosis. Cells were seeded in six-well plates and cultured overnight. Medium was replaced with serum-free medium and cells were treated with different concentrations of cisplatin for 24 h. Cell lysates were then subjected to Western blot.

Article Snippet: The antibodies used in this study includes COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), STAT6 for Western blot (Cell Signaling Technology, Danvers, MA, USA), for immunofluorescence (IF) (BD Pharmingen, San Diego, CA, USA), for ChIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-STAT6 (Cell Signaling Technology), p300 for Western blot, (Upstate, Lake Placid, NY, USA), for ChIP (Santa Cruz Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology), mouse immunoglobulin (Ig)G1, k Ig (BD Pharmingen), glyceraldehyde-3-phosphate dehydrogenase (Advanced ImmunoChemical, Long Beach, CA, USA), Cy3conjugated AffiniPure Goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA) and PARP (Cell Signaling Technology).

Techniques: Cell Culture, Western Blot

Journal: Antioxidants (Basel, Switzerland)

Article Title: Anti-Neuroinflammatory Effects of the Human Milk Oligosaccharide, 2'-Fucosyllactose, Exerted via Modulation of M2 Microglial Activation in a Mouse Model of Ischemia-Reperfusion Injury.

doi: 10.3390/antiox12061281

Figure Lengend Snippet: Figure 5. 2′-FL activates STAT6 after ischemic brain injury. (a) Representative Western blot and its densitometric analysis of (b) pSTAT6, (c) PGC1α, and (d) pAMPK in whole brain at 3 and 7 days after MCAO (n = 3). (e) Photomicrograph for pSTAT6+/Iba1+ in the striatum of ipsilateral side and (f) its histogram for IOD of pSTAT6+ cells (n = 3). (g) Level of IL-4 in the whole brain (n = 6). Data are expressed as Mean ± SEM. # < 0.05 vs. the Con group, * < 0.05, ** < 0.01 vs. the vehicle group. Scale bar = 100 µm.

Article Snippet: The antibodies against β-actin, NQO1, STAT6, and phospho-STAT6 (pSTAT6) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and the antibody against PGC1α was obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Western Blot

Journal: Antioxidants (Basel, Switzerland)

Article Title: Anti-Neuroinflammatory Effects of the Human Milk Oligosaccharide, 2'-Fucosyllactose, Exerted via Modulation of M2 Microglial Activation in a Mouse Model of Ischemia-Reperfusion Injury.

doi: 10.3390/antiox12061281

Figure Lengend Snippet: Figure 6. The proposed neuroinflammation mechanism of 2′-FL in regulating microglial M1/M2 polarization following ischemic injury via IL-4/STAT6 signaling pathways. Under ischemic injury conditions, STAT6 can be activated by IL-4 in the presence of 2′-FL, thereby regulating the expression of IL-10 and activating M2 microglial polarization. M2 activation leads to the inhibition of ROS generation through the downregulation of iNOS and the upregulation of HO1, NQO1, and SOD.

Article Snippet: The antibodies against β-actin, NQO1, STAT6, and phospho-STAT6 (pSTAT6) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and the antibody against PGC1α was obtained from Novus Biologicals (Centennial, CO, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Inhibition

Journal: Respiratory Research

Article Title: ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

doi: 10.1186/s12931-020-01568-w

Figure Lengend Snippet: Distinct expression profiles for receptors and epithelial progenitors in normal and hypoplastic lungs. a Examples of representative blots are shown for each protein analyzed. Western blot analysis of b ROBO1; c ROBO2; d SOX2; and e SOX9 relative expression levels in normal (ctrl) and hypoplastic (hyp) lungs at selected gestational ages from E13.5-to-E21.5. Each lane represents a pooled-tissue sample, and relative expression was determined against β-Tubulin. Semi-quantitative analysis of three independent experiments for each protein is plotted (n ≥ 9 per timepoint and experimental groups, respectively). Results are presented as mean ± SEM. Symbols indicate the main effects and non-redundant interactions of the two-way ANOVA. p < 0.05: α vs. ctrl; β vs. E13.5- and 15.5-ctrl; Ψ vs E13.5- and E15.5-hyp; γ vs E15.5-ctrl; µ vs E15.5-hyp; λ vs E17.5-ctrl; ε vs E17.5-hyp; δ vs E19.5-hyp

Article Snippet: Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 (1:500, ON, 4 oC; Cat No. sc25672, Santa Cruz Biotechnology Inc., USA), ROBO2 (1:250, ON, 4 oC; Cat No. sc16615, Santa Cruz Biotechnology Inc. USA), SOX2 (1:250, ON, 4 oC; Cat No. AF2018, R&D system, USA), SOX9 (1:250, ON, 4 oC; Cat No. AF3075, R&D system, USA), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (1∶5000; Cat No. #4270, Cell Signaling Technology Inc., USA), total β-Catenin (1∶30,000; Cat No. #NBP1-54,467, NOVUS Biologicals, USA), and BMP4 (1:250, ON, 4 oC; Cat No. sc-6896, Santa Cruz Biotechnology Inc., USA) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot

Journal: Respiratory Research

Article Title: ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

doi: 10.1186/s12931-020-01568-w

Figure Lengend Snippet: ROBO1 expression profile in normal and hypoplastic lungs. a representative immunohistochemical evidence for the presence of ROBO1 during normal ( aA–aH ) and hypoplastic (hyp, aa–ah ) fetal lung development at the distinct gestational ages. Symbols in each figure identify the distinct pulmonary structures: *bronchi; ¤primordia of bronchiole; ¥terminal bronchiole; {bronchioalveolar duct junction; &alveolar duct. Data is representative of n ≥ 3 independent experiments per stage/protein. b ROBO1 stained cells quantified by pulmonary structure and developmental stage. Results are presented as mean ± SEM. Symbols indicate the main effects and non-redundant interactions of one-way ANOVA. α p < 0.05. Scale bar 50 µm

Article Snippet: Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 (1:500, ON, 4 oC; Cat No. sc25672, Santa Cruz Biotechnology Inc., USA), ROBO2 (1:250, ON, 4 oC; Cat No. sc16615, Santa Cruz Biotechnology Inc. USA), SOX2 (1:250, ON, 4 oC; Cat No. AF2018, R&D system, USA), SOX9 (1:250, ON, 4 oC; Cat No. AF3075, R&D system, USA), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (1∶5000; Cat No. #4270, Cell Signaling Technology Inc., USA), total β-Catenin (1∶30,000; Cat No. #NBP1-54,467, NOVUS Biologicals, USA), and BMP4 (1:250, ON, 4 oC; Cat No. sc-6896, Santa Cruz Biotechnology Inc., USA) according to the manufacturer's instructions.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Respiratory Research

Article Title: ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

doi: 10.1186/s12931-020-01568-w

Figure Lengend Snippet: Effect of ROBO1 or ROBO2 functional impairment in branching morphogenesis. a The upper panel is representative of untreated lung explants (0 ng/mL) at D0; the bottom panel represents lung explants treated with recombinant IgG protein (0 ng/mL) and several doses of recombinant ROBO1 or ROBO2 proteins at day 4 (D4). b Morphometric analysis of the number of peripheral airway buds of fetal rat lung explants treated with increasing concentrations of recombinant ROBO1 (black) and ROBO2 (gray) proteins. Results are expressed as the D4/D0 ratio. c Examples of representative blots are showed for each analysed protein. Protein expression levels of d ROBO; e SOX2; f SOX9; g total β-Catenin; h non-phospho (active) β-Catenin; and i BMP4 in normal explant cultures treated with recombinant rat ROBO1 Fc Chimera for ROBO1 inhibition (black line) or recombinant human ROBO2 Fc Chimera for ROBO2 functional impairment (gray line). Recombinant human IgG Fc was used as control (IgG). n ≥ 9 per protein/condition. Each lane represents a pooled-tissue sample, and relative expression was determined against β-Tubulin and IgG. The data are presented as means ± SEM. p < 0.05: α vs. IgG

Article Snippet: Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 (1:500, ON, 4 oC; Cat No. sc25672, Santa Cruz Biotechnology Inc., USA), ROBO2 (1:250, ON, 4 oC; Cat No. sc16615, Santa Cruz Biotechnology Inc. USA), SOX2 (1:250, ON, 4 oC; Cat No. AF2018, R&D system, USA), SOX9 (1:250, ON, 4 oC; Cat No. AF3075, R&D system, USA), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (1∶5000; Cat No. #4270, Cell Signaling Technology Inc., USA), total β-Catenin (1∶30,000; Cat No. #NBP1-54,467, NOVUS Biologicals, USA), and BMP4 (1:250, ON, 4 oC; Cat No. sc-6896, Santa Cruz Biotechnology Inc., USA) according to the manufacturer's instructions.

Techniques: Functional Assay, Recombinant, Expressing, Inhibition, Control

Journal: Respiratory Research

Article Title: ROBO2 signaling in lung development regulates SOX2/SOX9 balance, branching morphogenesis and is dysregulated in nitrofen-induced congenital diaphragmatic hernia

doi: 10.1186/s12931-020-01568-w

Figure Lengend Snippet: a Overview of the main changes in spatiotemporal distribution of ROBO1, ROBO2, SOX2 and SOX9 at pseudoglandular (E17.5) and saccular stages (E21.5) in hypoplastic (hyp) fetal lungs (lower panel). w/o without b A proposed model of ROBO regulation of ex vivo branching morphogenesis through BMP4, β-Catenin, SOX2 and SOX9

Article Snippet: Blots were blocked in 5% bovine serum albumin and probed with primary antibodies to ROBO1 (1:500, ON, 4 oC; Cat No. sc25672, Santa Cruz Biotechnology Inc., USA), ROBO2 (1:250, ON, 4 oC; Cat No. sc16615, Santa Cruz Biotechnology Inc. USA), SOX2 (1:250, ON, 4 oC; Cat No. AF2018, R&D system, USA), SOX9 (1:250, ON, 4 oC; Cat No. AF3075, R&D system, USA), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (1∶5000; Cat No. #4270, Cell Signaling Technology Inc., USA), total β-Catenin (1∶30,000; Cat No. #NBP1-54,467, NOVUS Biologicals, USA), and BMP4 (1:250, ON, 4 oC; Cat No. sc-6896, Santa Cruz Biotechnology Inc., USA) according to the manufacturer's instructions.

Techniques: Ex Vivo

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion

doi: 10.1152/ajpgi.00219.2019

Figure Lengend Snippet: IL-4-mediated increase in transmembrane member 16A (TMEM16A) and phosphorylated STAT6 (pSTAT6) expression in cholangiocytes isolated from normal mouse liver. A: cell type-specific primers are used to amplify cDNA from cholangiocytes isolated from normal mouse liver. The hepatocyte cell line AML12 serves as a negative control and the TATA-binding protein (Tbp) as a positive control. Amplification of epithelial cell adhesion molecule (EpCAM) is specific to cholangiocytes, and albumin (Alb) is specific to hepatocytes. B: a representative Western blot of murine cholangiocytes showing TMEM16A protein expression levels in control conditions (nontreated) and after IL-4 treatment (40 ng/mL × 24 h). β-Actin was used as a loading control. A representative unedited figure is shown in Supplemental Fig. S4 (https://doi.org/10.35092/yhjc.11811741.v1). Cumulative data showing TMEM16A protein density before and after treatment with IL-4 are reported as % of control levels. Bars represent means ± SE; n = 3. *P < 0.05. C: a representative Western blot of murine cholangiocytes showing pSTAT6 and total STAT6 protein expression levels in control conditions (nontreated) and after IL-4 treatment (40 ng/mL × 24 h). β-Actin was used as a loading control. A representative unedited figure is shown in Supplemental Fig. S4. Cumulative data showing pSTAT6 and STAT6 protein density before and after treatment with IL-4 is reported as % of control levels. Bars represent means ± SE; n = 3. **P < 0.01.

Article Snippet: Total STAT-6 and phosphorylated STAT6 (p-STAT6) protein were detected in mouse liver and NRC cells by immunoblot utilizing rabbit monoclonal STAT6 (D3H4: no. 5397; Cell Signaling Technology, Danvers, MA), rabbit monoclonal pSTAT6 (D8S9Y, no. 56554; Cell Signaling Technology), or rabbit polyclonal pSTAT6 antibody (E-AB-20986; Elabscience, Houston, TX).

Techniques: Expressing, Isolation, Negative Control, Binding Assay, Positive Control, Amplification, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion

doi: 10.1152/ajpgi.00219.2019

Figure Lengend Snippet: IL-4-mediated increase in phosphorylated STAT6 (pSTAT6) and transmembrane member 16A (TMEM16A) expression in rat cholangiocytes is inhibited by leflunomide. A: a representative Western blot of normal rat cholangiocyte (NRC) lysates showing TMEM16A, pSTAT6, and total STAT6 expression levels in control conditions, after treatment with IL-4 (20 ng/mL × 24 h), and in the presence or absence of leflunomide (100 µM). β-Actin was used as a loading control. A representative unedited figure is shown in Supplemental Fig. S5 (https://doi.org/10.35092/yhjc.11811741.v1). B and C: cumulative data showing TMEM16A (B) and pSTAT6 and STAT6 (C) protein density before and after treatment with IL-4 in the presence or absence of leflunomide. Data reported as % of control levels. Bars represent means ± SE; n = 4 each; *P < 0.05 and **P < 0.01.

Article Snippet: Total STAT-6 and phosphorylated STAT6 (p-STAT6) protein were detected in mouse liver and NRC cells by immunoblot utilizing rabbit monoclonal STAT6 (D3H4: no. 5397; Cell Signaling Technology, Danvers, MA), rabbit monoclonal pSTAT6 (D8S9Y, no. 56554; Cell Signaling Technology), or rabbit polyclonal pSTAT6 antibody (E-AB-20986; Elabscience, Houston, TX).

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion

doi: 10.1152/ajpgi.00219.2019

Figure Lengend Snippet: IL-13-mediated increase in phosphorylated STAT6 (pSTAT6) and transmembrane member 16A (TMEM16A) expression in rat cholangiocytes is inhibited by leflunomide. A: a representative Western blot of normal rat cholangiocyte (NRC) lysates showing TMEM16A, pSTAT6, and total STAT6 expression levels in control conditions, after treatment with IL-13 (100 ng/mL × 24 h), and in the presence or absence of leflunomide (100 μM). β-Actin was used as a loading control. A representative unedited figure is shown in Supplemental Figure S5 (https://doi.org/10.35092/yhjc.11811741.v1). Cumulative data showing TMEM16A (B) and pSTAT6 and STAT6 (C) protein density before and after treatment with IL-13 in the presence or absence of leflunomide. Data reported as % of control levels. Bars represent means ± SE; n = 4 each. *P < 0.05 and **P < 0.01.

Article Snippet: Total STAT-6 and phosphorylated STAT6 (p-STAT6) protein were detected in mouse liver and NRC cells by immunoblot utilizing rabbit monoclonal STAT6 (D3H4: no. 5397; Cell Signaling Technology, Danvers, MA), rabbit monoclonal pSTAT6 (D8S9Y, no. 56554; Cell Signaling Technology), or rabbit polyclonal pSTAT6 antibody (E-AB-20986; Elabscience, Houston, TX).

Techniques: Expressing, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Signaling through the interleukin-4 and interleukin-13 receptor complexes regulates cholangiocyte TMEM16A expression and biliary secretion

doi: 10.1152/ajpgi.00219.2019

Figure Lengend Snippet: Inhibition of transmembrane member 16A (TMEM16A) or phosphorylated STAT6 (pSTAT6) abolishes the IL-13-mediated increase in transepithelial secretion in polarized normal rat cholangiocyte (NRC) monolayers. A: representative Ussing chamber studies demonstrating short-circuit current (Isc) measurements in response to ATP (100 μM) and the TMEM16A inhibitor T16Ainh-01 (10 μM) in control NRC (black) and after overnight treatment with IL-13 (100 ng/mL; blue). Reagents were added to the apical chamber at the time points indicated by the arrowheads. B: cumulative data showing average change in Isc (μA/cm2) in response to ATP (100 μM) in control (nontreated) and IL-13-treated monolayers and in the presence or absence of T16Ainh-01 (10 μM). The y-axis values are reported as ΔIsc (maximum Isc − basal Isc); n = 9–11 each. #P < 0.05, IL-13 treated vs. nontreated; *P < 0.05, T16Ainh-01 inhibits ATP-stimulated Isc. C: representative study of NRC monolayers incubated overnight with IL-13 (100 ng/mL) and mounted in Ussing chambers. Traces show short-circuit current (Isc) response in response to ATP (100 μM). Bottom trace: NRC preincubated with leflunomide (100 μM). D: cumulative data demonstrating ΔIsc (maximum Isc −basal Isc) in control NRC and after overnight treatment with IL-13 with and without leflunomide. Bars represent means ± SE; n = 6–7 each. *P < 0.05. IL-13 vs. control; **P < 0.05. leflunomide treated vs. untreated.

Article Snippet: Total STAT-6 and phosphorylated STAT6 (p-STAT6) protein were detected in mouse liver and NRC cells by immunoblot utilizing rabbit monoclonal STAT6 (D3H4: no. 5397; Cell Signaling Technology, Danvers, MA), rabbit monoclonal pSTAT6 (D8S9Y, no. 56554; Cell Signaling Technology), or rabbit polyclonal pSTAT6 antibody (E-AB-20986; Elabscience, Houston, TX).

Techniques: Inhibition, Incubation

Journal: Journal of Biological Chemistry

Article Title: Interleukin-4 Induces Senescence in Human Renal Carcinoma Cell Lines through STAT6 and p38 MAPK

doi: 10.1074/jbc.m113.499053

Figure Lengend Snippet: FIGURE 3. IL-4 activates STAT1, STAT3, and STAT6. A, Caki-1 cells were exposed to 10 ng/ml of IL-4 and analyzed for STAT phosphorylation by immunoblot- ting. B, Caki-1, A498, and 786-O cells were exposed to 1 ng/ml of IL-4, and STAT6 phosphorylation was analyzed by immunoblotting. C, Caki-1 cells were incubated with 1 ng/ml of IL-4, fixed, and immunostained with an anti-STAT6 antibody. D, Caki-1 cells were exposed to 10 ng/ml of IL-4 for 10 min, nuclear extracts were prepared, and EMSA was performed using a 32P-labeled STAT6 probe. Where indicated, anti-STAT1, -STAT3, or -STAT6 antibody was added for a supershift assay. The sequence specificity of the STAT6 binding was demonstrated by competition with a 50-fold excess of the cold STAT6 or AP-1 probe.

Article Snippet: For electrophoretic mobility shift assays (EMSA), STAT3 and STAT6 probes were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and NF- B and AP-1 probes were purchased from Promega (Madison, WI).

Techniques: Phospho-proteomics, Western Blot, Incubation, Labeling, Sequencing, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Interleukin-4 Induces Senescence in Human Renal Carcinoma Cell Lines through STAT6 and p38 MAPK

doi: 10.1074/jbc.m113.499053

Figure Lengend Snippet: FIGURE 4. Silencing STAT6 abrogates IL-4-induced growth inhibition. Caki-1 cells were transfected with control (scrambled, SC) or STAT6 (STAT6-1) siRNA. After 48 h, cells were exposed to 10 ng/ml of IL-4 for 16 h. A, STAT6 expression was analyzed by immunoblotting 48 h after siRNA transfection. B, incorporation of [3H]thymidine was measured using a liquid scintillation counter. **, p 0.01 (Student’s t test). The data shown are representative of three independent experiments.

Article Snippet: For electrophoretic mobility shift assays (EMSA), STAT3 and STAT6 probes were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and NF- B and AP-1 probes were purchased from Promega (Madison, WI).

Techniques: Inhibition, Transfection, Control, Expressing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Interleukin-4 Induces Senescence in Human Renal Carcinoma Cell Lines through STAT6 and p38 MAPK

doi: 10.1074/jbc.m113.499053

Figure Lengend Snippet: FIGURE 6. STAT6 and p38 MAPK independently mediate IL-4-induced growth inhibition and cellular senescence. A–C, Caki-1 cells were transfected with scrambled (SC), p38 MAPK or STAT6 (STAT6–1) siRNA. A, after 48 h, cells were serum-starved for 24 h and pretreated with SB203580 (20 M) for 1 h. Cells were theexposedto10ng/mlofIL-4for24h,anda[3H]thymidineincorporationassaywasperformed.B,5daysafterIL-4exposure,SA--galstainingwasperformed. More than 200 cells were counted in each of three randomly selected fields, and the percentage of SA--gal-positive cells was plotted. C, 16 h after IL-4 exposure (10 ng/ml), cells were harvested for an immunoblot analysis. D, the control pGL4 and p4xSTAT6-Luc2P plasmids were transfected into Caki-1 cells. After 48 h, cells were split and transfected again with scrambled (SC), p38, and/or STAT6 siRNAs for 24 h. Cells were serum-starved for 24 h and incubated with 20 ng/ml of IL-4 for 6 h. Then a luciferase assay was performed with the cell lysates. The data shown are mean S.D. of three independent experiments. RLU, relative luciferase units. E, Caki-1 cells were pretreated with 20 M SB203580 for 1 h and subsequently exposed to 1 ng/ml of IL-4 for 1 h. Total cell lysates were subjected to an immunoblot analysis with anti-STAT6, anti-p-STAT6, and anti-tubulin antibodies. F, Caki-1 cells were transfected with an empty vector, pcDNA3-FLAG-STAT6 (STAT6 or FL-STAT6), and/or pcDNA3-p38 MAPK (p38) as indicated. After 3 days, SA--gal staining was performed (left panel). Protein expression of FLAG-STAT6 and p38 MAPK was assessed by an immunoblot analysis (right panel).

Article Snippet: For electrophoretic mobility shift assays (EMSA), STAT3 and STAT6 probes were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and NF- B and AP-1 probes were purchased from Promega (Madison, WI).

Techniques: Inhibition, Transfection, Western Blot, Control, Incubation, Luciferase, Plasmid Preparation, Staining, Expressing

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) Expression of STAT6 but not STAT3 was dramatically reduced during KSHV reactivation. Whole cell lysates from KSHV-infected BCBL1 and BC3 and uninfected BJAB cells individually treated with or without 20 ng/ml of TPA and 1.5mM sodium butyrate (T/NB) for 24 h, were subjected to immunoblotting (IB) with antibodies as indicated in the figure. The relative density (RD) of STAT6 protein band was quantitated. ( B ) Early infection of KSHV reduced STAT6 expression. Whole cell lysate from HUVEC cells with KSHV infection (GFP positive) at different time points, were subjected to immunoblotting (IB) with antibodies as indicated in the figure. ( C ) Doxycycloline (Dox)-induced RTA expression in iSLK-RTA or iSLK-219 cells led to the decreased expression of STAT6 but not of STAT3. Whole cell lysates from the different-induction time points were subjected to immunoblotting (IB) with antibodies as indicated in the figure. ( D ) Quantitative PCR analysis of transcriptional level of STAT6 and STAT3 in iSLK-RTA cells with doxycline induction. ( E ) Quantitative PCR analysis of STAT6 and RTA mRNA transcripts in BJAB, BCBL1 and BC3 cells treated with TPA and sodium butyrate (T/NB) for 0, 12 and 24 h. The relative level of mRNA transcript was present. Beta actin was used as internal control. ( F ) Reporter assays of STAT6 promoter. HEK293 cells co-transfected STAT6 promoter-driven luciferase reporter with different dosage RTA (0, 1, 5, 10μg) were subjected to reporter assay. Relative firefly luciferase unit (RLU) normalization with Renilla activity was analyzed. Data is presented as means±SD of three independent experiments. The expression of exogenous RTA was verified by immunoblotting assays and shown in the middle panel. Schematic of putative RBP-Jκ-binding sites within STAT6 promoter is shown at the bottom panel. ( G ) RTA reduces the protein stability of STAT6. HEK293T cells were co-transfected by FLAG-STAT6 with RTA-myc or vector alone. At 36 h post-transfection, cells were treated with Cycloheximide (CHX) 200μg/ml for the indicated time before harvesting and lysing for immunoblotting. The relative density (RD) of protein level of STAT6 is quantified based on triplicate experiments and shown at the bottom panel.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Expressing, Infection, Western Blot, Real-time Polymerase Chain Reaction, Control, Transfection, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Plasmid Preparation

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) 293T cells were transfected with exogenous FLAG-STAT6 in the presence or absence of myc-tagged RTA. At 24 h post-transfection, cells were individually subjected to treatment with lysosomal inhibitor Chloroquine (Chl) and 3-Methyladenine (3-MA), or proteasomal inhibitor MG-132, followed by immunoblotting with antibodies as indicated in the figure. The relative density (RD) of STAT6 protein band was quantitated. ( B ) RTA induced exogenous STAT6 co-localization with LC3B. 293T cells transfected with FLAG-STAT6, RFP-RTA and GFP-LC3B were subjected to immunofluorescent confocal assays with GFP (green), RFP (red) and FLAG (blue) antibody. Arrow indicates the co-localization of STAT6 with LC3B. ( C ) RTA induced endogenous STAT6 co-localization with LC3B. iSLK-RTA cells treated with or without doxycycloline (Dox) for 12 h, were subjected to immunofluorescent assays with antibodies against STAT6 or LC3B. Nuclei were stained with DAPI. ( D ) Inhibitors of lysosome and proteasome activity blocked RTA-induced STAT6 degradation during lytic reactivation. BCBL1 cells were induced with or without TPA and sodium butyrate (T/NB) for 12 h, followed by individually treatment with 3-MA, MG-132, or DMSO for 3 hours, and subjected to immunoblotting assays as indicated in figure.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Transfection, Western Blot, Staining, Activity Assay

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) Ectopic expression of exogenous STAT6 interacted with RTA. Whole cell lysate (WCL) from 293T cells co-transfected with different combination of FLAG-STAT6 and RTA-myc as indicated, were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. ( B ) Endogenous STAT6 associated with RTA in KSHV-positive cells with lytic reactivation. Whole cell lysate (WCL) from BC3 cells treated with 20 ng/ml of TPA and 1.5mM sodium butyrate (T/NB) or iSLK-219 cells treated with 2μg/ml doxycycloline (Dox) for 24h were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. ( C ) RTA associated with the carboxyl terminus of STAT6. 293T cells were transfected with expressing plasmids for 48 h, and the whole cell lysates (WCL) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. Schematic of STAT6 amino acid sequence and its truncation mutants were shown at the bottom panels. The red circle indicates the RTA-associated domains. The nuclear localization sequence (NLS), ubiquitylation (Ub) and phosphorylation (P) sites are shown.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Expressing, Transfection, Immunoprecipitation, Western Blot, Sequencing, Phospho-proteomics

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) Degradation of STAT6 induced by RTA relied upon its transactivation domain. 293T cells were transfected with expressing plasmids for 48 hours, and the whole cell lysates (WCL) were subjected to immunoprecipitation (IP) followed by immunoblotting (IB) or directly immunoblotting assays as indicated in figure. RD, relative density. ( B ) Endogenous phosphorylated STAT6 on Y641 was not significantly reduced by reactivation of the lytic cycle in PEL cells. BCBL1 cells treated with or without TPA and sodium butyrate (NaB) were subjected to immunoblotting assays as indicated in figure. ( C ) RTA did not block IL-4 induction of STAT6 phosphorylation on Y641. 293T cells were transfected with exogenous FLAG-STAT6 in the presence or absence of myc-tagged RTA. At 24 h post-transfection, cells were subjected to treatment with 20nM IL-4 for 6h before harvesting for immunoblotting with antibodies as indicated in the figure. ( D ) RTA-induced degradation of STAT6 was dependent on its RING-domain. HEK293T cells were transfected STAT6 with FLAG tag in the presence or absence of wild type (WT) RTA with myc tag or its mutants for 48 h, and subjected to immunoblotting (IB) with antibodies as indicated in the figure. The relative density (RD) of STAT6 protein band was quantitated. Brief schematic of RTA amino acid sequence and its mutants is shown on the right panel.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Blocking Assay, Phospho-proteomics, FLAG-tag, Sequencing

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) RTA induced STAT6 ubiquitylation. HEK293T cells were co-transfected with different expressing plasmids as indicated. At 36 h post-transfection, cells were treated with proteasomal inhibitor MG132 for 6 h before harvesting and lysing for immunoprecipitation (IP) and immunoblotting (IB). ( B ) K48 and K63-linked ubiquitylation of exogenous STAT6 was induced by RTA. HEK293T cells were co-transfected and treated as in panel A. HA-tagged wild type (WT) ubiquitin and its lysine mutants containing only K6, K48 or K63 were used. ( C ) K48 and K63-linked ubiquitylation of endogenous STAT6 was significantly induced during KSHV reactivation. BCBL1 cells were treated with TPA and sodium butyrate (T/NB) for 24 h, followed by MG132 treatment for 8 h before harvesting. Whole cell lysates (WCLs) were lysed for denatured immunoprecipitation (IP) using antibodies specific against K48 or K63 polyubiquitin and immunoblotting (IB) as indicated in the figure.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) The expression of 49 cellular genes was significantly affected by STAT6 knockdown in PEL cells. BC3 and BCBL1 cells with or without STAT6 knockdown were individually subjected to RNA deep-sequencing analysis. The cellular genes with significantly change in the presence of STAT6 knockdown are shown. ( B ) Reduction of STAT6 led to the increased gene expression of MHC II-, cytokine- or anti-apoptosis-related proteins in PEL cells. The molecules related to antigen processing and presentation, cell migration, tumor suppressor, and chromosome stability are highlighted. ( C ) Expression of TRIML2 and AIM1 was consistently enhanced by RTA or STAT6 knockdown. The results were the RNA deep-sequencing analysis of iSLK cells with Doxycycline-inducible RTA and PEL cells with STAT6 knockdown. ( D ) Quantitative PCR analysis of TRIML2, AIM1 and CIITA expression in the iSLK-RTA and iSLK-219 cells treated with Doxycycline, or BCBL1 cells treated with TPA and sodium butyrate (T/NB) for 24 h.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Expressing, Knockdown, Sequencing, Gene Expression, Migration, Real-time Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus

doi: 10.1371/journal.ppat.1007416

Figure Lengend Snippet: ( A ) EBV reactivation led to inhibition of STAT6 expression and increased monoubiquinated TRIML2. Whole cell lysates from EBV-positive (LCL and B95.8) and negative (DG75) B lymphoma cells with TPA and sodium butyrate treatment for 24 h, were individually subjected to immunoblotting as indicated in the figure. ( B ) and ( C ) De novo infection of HCMV and HSV1 led to inhibition of STAT6 expression and increased monoubiquinated TRIML2 in early lytic replication. Mrc-5 (human fetal lung fibroblast cells) and Vero (monkey kidney epithelial cells) were individually infected with HCMV and HSV1 at an MOI of 3 and 0.1 as described in material and method. Cells were harvested at different time points and lysed for immunoblotting as indicated in the figure. RD, relative density. ( D ) Proposed model of RTA-induced degradation of STAT6 upon KSHV reactivation. During reactivation of KSHV latently-infected cells by stimuli like TPA and sodium butyrate, KSHV encoded RTA not only blocks formation of LANA-RBP-Jκ complex (Lan et al, J Virol 2004, 2005), but also induces K48- and K63-linked ubiquitylation of STAT6 for degradation via proteasome and lysosome. The interaction between STAT6 and RTA not only leads to STAT6 degradation but also increase TRIML2 expression and ubiquitylation, which in turn prolongs host cell survival in lytic replication and enhances RTA expression, and facilitates viral progeny production. Similar effects are observed in other human herpesviruses including EBV, HCMV and HSV1 as indicated.

Article Snippet: Antibodies to STAT6 (D3H4, Cell Signaling technology), p-STAT6 (Tyr-641, Cell Signaling Technology), FLAG (M2, Sigma), p53 (Do-1, Santa cruz), PARP1 (F2, Santa cruz), Caspase-3 (H-277, Santa cruz), TRIML2 (ARP55636-P050, Aviva System Biology), EBV ZEBRA (sc-53904, Santa cruz), ICP0 (11060, Santa cruz), Ub-K48 (05–1307, EMD Millipore),Ub-K63 (05–1308, EMD Millipore), LC3B (GT3612,GeneTex) and GAPDH (G8140-01, US Biological) were used according to the manufacturers specifications.

Techniques: Inhibition, Expressing, Western Blot, Infection

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of ET-1 on Tyr and Ser phosphorylation of STAT3 in cultured rat astrocytes. A, time course. Serum-starved astrocytes were treated with 100 nm ET-1 for the time indicated. Tyr-705– and Ser-727–phosphorylated STAT3 were detected by immunoblotting. After detection of phosphorylated proteins, the same blots were reprobed with an anti-STAT3 antibody to detect levels of total STAT3. The protein bands in X-ray films were subjected to densitometry analyses. Results are means ± S.D. (error bars) of four experiments. Individual data points are indicated to the right of the error bars. STAT3 phosphorylation values are presented as ratios of phosphorylated/total STAT3. ●, phospho-Tyr STAT3; ○, phospho-Ser STAT3. Expression levels of total STAT3 are presented as a ratio to β-actin protein. *, p < 0.05; **, p < 0.01 versus zero time by one-way ANOVA followed by Dunnett's test. B, dose-response. Astrocytes were treated with the indicated concentrations of ET-1 for 20 min. Results are the means ± S.D. of 4–8 experiments and presented as ratios of Ser-phosphorylated/total STAT3. Individual data points are indicated by dots on the error bars. **, p < 0.01 versus none by one-way ANOVA followed by Dunnett's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Cell Culture, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: A, effects of Ala1,3,11,15-ET-1 on Ser-727 phosphorylation of astrocytic STAT3. Serum-starved astrocytes were treated with 100 nm Ala1,3,11,15-ET-1 for the time indicated. Results are means ± S.D. (error bars) of four experiments and are presented as ratios of Ser-phosphorylated/total STAT3. Individual data points are indicated to the right of the error bars. *, p < 0.05 versus zero time by one-way ANOVA followed by Dunnett's test. B, effects of ETB receptor agonist on the ET-induced Ser phosphorylation of STAT3. Cultured astrocytes were treated with 100 nm ET-1 for the time indicated. After detection of Ser-727–phosphorylated STAT3, levels of total STAT3 were measured. Results are means ± S.D. of six experiments. Individual data points are indicated by dots on the error bars. **, p < 0.01 versus none without ET antagonist; ##, p < 0.01 versus ET-1 without ET antagonist by one-way ANOVA followed by Tukey's test. NS, not significant.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: A and B, immunocytochemical observations of phosphorylated STAT3 in cultured astrocytes. Serum-starved astrocytes were treated with 100 nm ET-1 for 30 min. After fixation, cells were labeled with anti-phospho-Tyr (A) or anti-phospho-Ser (B) STAT3 antibody. For counterstaining, PI was included in the secondary incubations with an FITC-conjugated secondary antibody. Typical micrographs of phospho-STAT3–positive astrocytes treated with ET-1 are shown. Phospho-STAT3 immunoreactivity was observed in the nucleus (arrowheads). Bar, 50 μm. C, increases in DNA binding activities of STAT3 to consensus oligonucleotides by ET-1. Cultured astrocytes were treated with 100 nm ET-1 for 30 and 60 min. After nucleus extracts were obtained from cultured astrocytes, the binding activity to STAT3 consensus DNA sequence was measured using an ELISA-based kit. Results are expressed as the mean ± S.D. (error bars) of 7–8 experiments. Individual data points are indicated by dots on the error bars. **, p < 0.01 versus none by one-way ANOVA followed by Dunnett's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Cell Culture, Labeling, Binding Assay, Activity Assay, Sequencing, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of inhibitors for PKC, ERK, JAK, and Src on the ET-induced Ser phosphorylation of STAT3. Astrocytes were treated with 100 nm ET-1 for 20 min. Go6983 (1 μm), FR180204 (5 μm), ruxolitinib (500 nm), or PP-1 (1 μm) was added to the medium 30 min before treatment with ET-1. Results are means ± S.D. (error bars) of 7–8 experiments and are presented as ratios of Ser-phosphorylated/total STAT3. Individual data points are indicated by dots on the error bars. **, p < 0.01 versus none; #, p < 0.05 versus ET-1 by one-way ANOVA followed by Tukey's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of BQ788 administration on FPI-induced STAT3 phosphorylation in the moue cerebrum. BQ788 (15 nmol/day) or vehicle was repeatedly administered into the mouse brain from 2 days after FPI. Expression levels of phosphorylated and total STAT3 proteins in the injured areas of the cerebrum were measured 5 days after FPI. Typical immunoblots are indicated in the top left of the quantitative results. Expression levels of phosphorylated STAT3 were normalized to that of total STAT3. Expression levels of total STAT3 protein were normalized to that of β-actin. Results are mean ± S.D. (error bars) for four mice expressed as percentages of sham/vehicle. Individual data points are indicated by dots on the error bars. *, p < 0.05 versus sham/vehicle; #, p < 0.05; ##, p < 0.01 versus FPI/vehicle by one-way ANOVA with Tukey's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of STAT3 inhibition on ET-induced G1/S phase transition of cultured astrocytes. A and B, effects of STAT3 inhibitors. In the presence or absence of STAT3 inhibitors (5 μm stattic and 5 μm 5,15-DPP), serum-starved astrocytes were cultured with 100 nm ET-1 and 100 μm BrdU for 48 h. After fixation, cells were labeled with anti-BrdU mouse antibody. For counterstaining, PI was included in the secondary incubations with an FITC-conjugated anti-mouse IgG antibody. Cells showing BrdU reactivity in the nucleus were defined as BrdU-positive cells. A, typical micrograph of BrdU-positive astrocytes treated with ET-1. Arrowheads indicate BrdU reactivity in the nucleus. The panels on the right show PI staining in the same fields as those observed for BrdU staining. Bar, 50 μm. B, the numbers of BrdU-positive cells under each condition are expressed as percentages of the total number of PI-positive cells observed. Results are expressed as means ± S.D. (error bars) of 4–9 experiments, with more than 400 PI-positive cells observed in each experiment. Individual data points are indicated by dots on the error bars. **, p < 0.01 versus none without STAT3 inhibitors; ##, p < 0.01 versus ET-1 without STAT3 inhibitors by one-way ANOVA followed by Tukey's test. NS, not significant. C–E, effects of STAT3 siRNA. Knockdown of astrocytic STAT3 by siRNA was performed as described under “Experimental procedures.” C, immunoblot analysis showed that transfection of STAT3 siRNA decreased STAT3 in cultured astrocytes. An immunoblot image of STAT3 and β-actin in two independent transfections is shown. D, typical micrograph of BrdU-positive cells treated with ET-1 after siRNA transfection. Bar, 50 μm. E, the numbers of BrdU-positive cells under each condition are expressed as percentages of the total number of PI-positive cells observed. Results are expressed as means ± S.D. of six experiments, with more than 400 PI-positive cells observed in each experiment. Individual data points are indicated by dots on the error bars. *, p < 0.05; **, p < 0.01 versus none; ##, p < 0.01 versus control siRNA by one-way ANOVA followed by Tukey's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Inhibition, Sublimation, Cell Culture, Labeling, Staining, BrdU Staining, Western Blot, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of STAT3 inhibitors on expression of cyclin D1, SKP2, and p27 in cultured astrocytes. A, serum-starved astrocytes were treated with 100 nm ET-1 for 6 h. Stattic (5 μm) and 5,15-DPP (5 μm) were included in the medium at 30 min before the addition of ET-1. The expression of cyclin D1, SKP2, and p27 mRNAs was normalized to that of G3PDH. Results are expressed as means ± S.D. (error bars) of 8–13 experiments. Individual data points are indicated by dots on the error bars. *, p < 0.05; **, p < 0.01 versus none without STAT3 inhibitors; #, p < 0.05; ##, p < 0.01 versus ET-1 without STAT3 inhibitors by one-way ANOVA followed by Tukey's test. B, astrocytes were treated with 100 nm ET-1 for 16 h. Stattic (5 μm) and 5,15-DPP (5 μm) were included in the medium at 30 min before the addition of ET-1. The expression of cyclin D1 and SKP2 proteins was normalized to that of β-actin. Results are expressed as means ± S.D. of four experiments. Individual data points are indicated by dots on the error bars. **, p < 0.01 versus none without STAT3 inhibitors; #, p < 0.05; ##, p < 0.01 versus ET-1 without STAT3 inhibitors by one-way ANOVA followed by Tukey's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Expressing, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of STAT3 siRNA on expression of cyclin D1, SKP2, and p27 in cultured astrocytes. A, after transfection of STAT3 siRNA, 100 nm ET-1 was included in serum-free MEM, and cultured astrocytes were incubated for 3 h (SKP2) or 6 h (cyclin D1 and p27). The expression of cyclin D1, SKP2, and p27 mRNAs was normalized to that of G3PDH. Results are expressed as means ± S.D. (error bars) of 8–12 experiments. Individual data points are indicated by dots on the error bars. *, p < 0.05; **, p < 0.01 versus no ET-1. #, p < 0.05 versus control siRNA by one-way ANOVA followed by Tukey's test. B, after transfection of STAT3 siRNA, astrocytes were treated with 100 nm ET-1 for 16 h. Then cell lysate was prepared. The expression of cyclin D1 and SKP2 proteins was normalized to that of β-actin. Results are expressed as means ± S.D. of 4–6 experiments. Individual data points are indicated by dots on the error bars. *, p < 0.05; ***, p < 0.001 versus no ET-1. #, p < 0.05; ###, p < 0.001 versus control siRNA by one-way ANOVA followed by Tukey's test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Expressing, Cell Culture, Transfection, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Endothelin-1 stimulates expression of cyclin D1 and S-phase kinase–associated protein 2 by activating the transcription factor STAT3 in cultured rat astrocytes

doi: 10.1074/jbc.RA118.005614

Figure Lengend Snippet: Effects of ET-1 on STAT3 binding to 5′-flanking regions of cyclin D1 and SKP2 genes, as determined by ChIP-PCR assay. A, association of STAT3 protein with 5′-flanking regions of rat cyclin D1 and SKP2 genes. Nuclear extract from cultured astrocytes was subjected to ChIP with anti-STAT3 rabbit antibody. In place of anti-STAT3 antibody, nonimmune rabbit IgG and anti-histone H3 rabbit antibody were included in ChIP mixture as negative and positive controls, respectively. DNA fragments were extracted from the immunoprecipitants and amplified by PCR using primer pairs to detect 5′-flanking regions of rat cyclin D1 and SKP2 genes. DNA fragments extracted from nuclear preparation before ChIP (input DNA) or H2O (blank) were also subjected to PCR. The PCR products were electrophoresed in 2% agarose gel and stained with 1 μg/ml ethidium bromide. B, effects of ET-1 on STAT3 binding. After cultured astrocytes were treated with 100 nm ET-1 for 3 h, nuclear extract for ChIP-PCR was prepared. After ChIP using an anti-STAT3 antibody, DNA fragments were extracted from the STAT3 immunoprecipitants. DNA fragments of the rat cyclin D1 or SKP2 gene 5′-flanking region in STAT3 immunoprecipitants were quantified by quantitative PCR. Results are expressed as means ± S.D. (error bars) of six experiments. Individual data points are indicated by dots on the error bars. *, p < 0.05 versus none by Student's t test.

Article Snippet: For negative or positive control, an equal amount of nonimmune rabbit IgG (Cell Signaling Technology, catalog no. 2729) or anti-histone H3 rabbit antibodies (Cell Signaling Technology, catalog no. 4620) was included in the ChIP mixture instead of anti-STAT3 antibody.

Techniques: Binding Assay, Cell Culture, Amplification, Agarose Gel Electrophoresis, Staining, Real-time Polymerase Chain Reaction