Journal: The international journal of biochemistry & cell biology

Article Title: Depletion of Cellular Glutathione Modulates LIF-Induced JAK1-STAT3 Signaling in Cardiac Myocytes

doi: 10.1016/j.biocel.2012.08.016

Figure Lengend Snippet: GSH depletion inhibits LIF-induced activation of JAK1 and evidence JAK1 is redox-sensitive. (A) Cardiac myocytes were treated 6 h with various concentrations of BSO (lanes 3-6), washed 2× with medium, and incubated 24 h in serum-free medium. Following treatment for 10 min with vehicle (lane 1) or 2 ng/mL LIF (lanes 2-6), cell lysates were prepared. JAK1 in lysates was immunoprecipitated and analyzed for tyrosine phosphorylation (pY) by Western immunoblotting. Membranes were stripped and reprobed for total JAK1 as a loading control. Graph shows densitometric analysis of immunoreactive bands. Values are mean ± SE of 4 independent experiments. **P < 0.01 and ***P < 0.001 vs. LIF alone. (B) Oxidant exposure increases sulfenic acid formation in purified recombinant JAK1. Purified recombinant JAK1 was immunoprecipitated and treated with either 10 mM DTT, 2.5 mM o-IBZ or 60 μM H2O2 for 1 hr at 4°C, then processed as described under “Materials and Methods” to determine the sulfenic acid content within JAK1. Levels of cysteine-sulfenic acid and JAK1 were quantified by the Li-COR Odyssey Detection System. Treatment with o-IBZ or H2O2 resulted in a significant increase in relative sulfenic acid content. *P < 0.05 and ***P < 0.001 vs. DTT, n = 4.

Article Snippet: Sources for additional reagents used for sulfenic acid detection were as follows: Odyssey blocking buffer, nitrocellulose membranes, molecular weight marker, and IRDye secondary antibodies were from LI-COR Biosciences (Lincoln, NE); human recombinant JAK1 was from OriGene (Rockville, MD); anti-human JAK1 (anti-hJAK1) antibody was from BD Biosciences (Bedford, MA); and anti-cysteine-sulfenic acid antibody was from Millipore.

Techniques: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Purification, Recombinant

Journal: The international journal of biochemistry & cell biology

Article Title: Depletion of Cellular Glutathione Modulates LIF-Induced JAK1-STAT3 Signaling in Cardiac Myocytes

doi: 10.1016/j.biocel.2012.08.016

Figure Lengend Snippet: Replenishment of intracellular GSH reverses the inhibitory effect of GSH depletion on LIF-induced STAT3 and JAK1 activation. Cardiac myocytes were treated for 6 h with different concentrations of BSO (lanes 3,4,7,8), washed 2× with medium, and incubated for 24 h in serum-free medium containing vehicle or 2 mM glutathione monoethyl ester (GME) (lanes 5-8). Cells were dosed with vehicle (lanes 1 & 5) or 2 ng/mL LIF (lanes 2-4 & 6-8) for 10 min. (A) Cell lysates were prepared and analyzed by Western immunoblotting for STAT3 Y705 phosphorylation. Membranes were stripped and reprobed for total STAT3 and GAPDH to confirm equal loading. (B) Immunoprecipitates of JAK1 from cell lysates were immunoblotted for tyrosine phosphorylated JAK1 (upper) and total JAK1 (lower) to ensure equal loading. Blots shown are representative of 5 independent experiments. (C) Densitometric analysis of immunoreactive bands. *P < 0.05 and ***P < 0.001 vs. maximal activation with LIF, ANOVA and Dunnett’s multiple comparison test.

Article Snippet: Sources for additional reagents used for sulfenic acid detection were as follows: Odyssey blocking buffer, nitrocellulose membranes, molecular weight marker, and IRDye secondary antibodies were from LI-COR Biosciences (Lincoln, NE); human recombinant JAK1 was from OriGene (Rockville, MD); anti-human JAK1 (anti-hJAK1) antibody was from BD Biosciences (Bedford, MA); and anti-cysteine-sulfenic acid antibody was from Millipore.

Techniques: Activation Assay, Incubation, Western Blot

Journal: The international journal of biochemistry & cell biology

Article Title: Depletion of Cellular Glutathione Modulates LIF-Induced JAK1-STAT3 Signaling in Cardiac Myocytes

doi: 10.1016/j.biocel.2012.08.016

Figure Lengend Snippet: N-Acetylcysteine (NAC) prevents the inhibitory effect of GSH depletion on LIF-induced STAT3 and JAK1 activation. Cardiac myocytes were treated for 6 h with different concentrations of BSO, washed 2× with medium, and incubated for 24 h in serum-free medium containing vehicle or NAC (10 mM). Cells were dosed for 10 min with vehicle (lane 1) or 2 ng/mL LIF (lanes 2 - 7). (A) Western immunoblots of cell lysates were sequentially probed for STAT3 Y705 phosphorylation, STAT3, and GAPDH (equal loading control). (B) Immunoprecipitated JAK1 from cell lysates was resolved by SDS-PAGE and the blots sequentially probed for phosphotyrosine and total JAK1 protein. The blots shown are representative of 4 and 3 independent experiments for A and B, respectively. (C) Densitometric analysis of immunoreactive bands. *P < 0.05 vs. maximal activation with LIF, ANOVA and Dunnett’s multiple comparison test (top panel). *P < 0.05 and **P < 0.01 noted comparisons and †P < 0.05 vs. LIF, ANOVA and Newman–Keuls post-test (bottom panel).

Article Snippet: Sources for additional reagents used for sulfenic acid detection were as follows: Odyssey blocking buffer, nitrocellulose membranes, molecular weight marker, and IRDye secondary antibodies were from LI-COR Biosciences (Lincoln, NE); human recombinant JAK1 was from OriGene (Rockville, MD); anti-human JAK1 (anti-hJAK1) antibody was from BD Biosciences (Bedford, MA); and anti-cysteine-sulfenic acid antibody was from Millipore.

Techniques: Activation Assay, Incubation, Western Blot, Immunoprecipitation, SDS Page

Journal: The international journal of biochemistry & cell biology

Article Title: Depletion of Cellular Glutathione Modulates LIF-Induced JAK1-STAT3 Signaling in Cardiac Myocytes

doi: 10.1016/j.biocel.2012.08.016

Figure Lengend Snippet: Differential response of STAT1 and STAT3 to crosslinking agents. (A & B) Aliquots of mouse heart homogenates were incubated with vehicle, 500 μM NCP, 1 mM diamide, or 500 μM NCP + 1 mM diamide for 30 min. Samples were processed for SDS-PAGE and Western analysis in nonreducing or reducing sample buffer. (A) Membranes were probed for STAT3 and STAT1 using the Li-COR Odyssey detection system. (B) For STAT1 and STAT3, the intensity of the band in the nonreduced sample was normalized to the intensity of the band after reduction and expressed relative to the total oxidized sample (NCP + diamide). ***P < 0.001 vs. Control STAT3, 2-way ANOVA and Bonferroni post-test (n = 3 mouse hearts). (C) Proposed scheme based on our findings and the literature to explain the accumulating evidence that oxidative stress attenuates gp130 cytokine signaling by targeting JAK1/2 and STAT3.

Article Snippet: Sources for additional reagents used for sulfenic acid detection were as follows: Odyssey blocking buffer, nitrocellulose membranes, molecular weight marker, and IRDye secondary antibodies were from LI-COR Biosciences (Lincoln, NE); human recombinant JAK1 was from OriGene (Rockville, MD); anti-human JAK1 (anti-hJAK1) antibody was from BD Biosciences (Bedford, MA); and anti-cysteine-sulfenic acid antibody was from Millipore.

Techniques: Incubation, SDS Page, Western Blot

Journal: Frontiers in Immunology

Article Title: Loss of Janus Associated Kinase 1 Alters Urothelial Cell Function and Facilitates the Development of Bladder Cancer

doi: 10.3389/fimmu.2019.02065

Figure Lengend Snippet: STAT1 phosphorylation and expression of IRF1 mRNA is impaired in JAK1-deficient hTERT urothelial cells. (A) JAK1 protein (InterPro P23458) structure showing core domains, TCGA BLCA variants and their predicted impact. Protein domains: FERM (4.1 protein, ezrin, radizin, moesin domain), PHD (pleckstrin homology-like domain), SH2 (Src homology 2), S-TY-PK (unknown specificity serine-threonine/tyrosine protein kinase), SY-PK (serine-tyrosine protein kinase). Variants: non-sense (red cross), missense (yellow inverted triangle), synonymous (green triangle), frameshift (purple square). Variant effects: S (SIFT score < 0.05), P (Poly-Phen score > 0.908), p (Poly-Phen score > 0.446 & ≤ 0.908), H (HMMvar score > 2). A single 5′ UTR modifier mutation not shown. (B,C) Analysis of JAK/STAT signaling by flow cytometry in untransduced (Unt), Sc and KD hTERT immortalized urothelial cell lines after stimulation with IFNγ (1 ng/ml) for 24 h. Data (A) is from a representative experiment, data (B) is from three independent experiments. Two-tailed Mann Whitney test. (D) RTqPCR analysis of IRF1 mRNA expression from KD and Sc hTERT immortalized urothelial cell lines after stimulation with IFNγ (1 ng/ml). Data is from four independent experiments. Two-tailed Mann Whitney test. * P < 0.05 Error bars represent the SE. (E,F) Flow cytometry analysis of IFNGR expression in KD and Sc hTERT immortalized urothelial cell lines following stimulation with IFNγ (5 ng/ml) for 2 days. The graph shows mean values ± SD. Data is from three independent experiments. One-way ANOVA with Tukey's Multiple Comparison Test.

Article Snippet: Mutations and their effects were presented against the JAK1 protein sequence (InterPro P23458).

Techniques: Phospho-proteomics, Expressing, Variant Assay, Mutagenesis, Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Frontiers in Immunology

Article Title: Loss of Janus Associated Kinase 1 Alters Urothelial Cell Function and Facilitates the Development of Bladder Cancer

doi: 10.3389/fimmu.2019.02065

Figure Lengend Snippet: JAK1-deficient hTERT urothelial cells demonstrate preserved population growth and reduced apoptosis in response to IFNγ. (A) KD and Sc hTERT immortalized urothelial cell lines were cultured with addition of Alamar Blue (AB) dye and stimulated with IFNγ (5 ng/ml) for the given time points. The capacity of viable cells to reduce AB dye was used as a proxy for cell number. Data (A) is from five independent experiments. (B–E) KD and Sc hTERT urothelial cell lines were stimulated with the given concentrations of IFNγ for 5 days. Percentage of early and late apoptosis was quantified with Annexin V/PI apoptosis detection kit by flow cytometry. Data (B–D) are from five independent experiments, data (E) is from a representative experiment. One-way ANOVA with Tukey's Multiple Comparison post-test. * P < 0.05. Error bars represent the SE.

Article Snippet: Mutations and their effects were presented against the JAK1 protein sequence (InterPro P23458).

Techniques: Cell Culture, Flow Cytometry, Comparison

Journal: Frontiers in Immunology

Article Title: Loss of Janus Associated Kinase 1 Alters Urothelial Cell Function and Facilitates the Development of Bladder Cancer

doi: 10.3389/fimmu.2019.02065

Figure Lengend Snippet: JAK1-deficient hTERT urothelial cells showed reduced lymphocyte-mediated killing in response to IFNγ. JAK1-deficient and Sc hTERT immortalized urothelial cell lines were pretreated or not with IFNγ and cultured overnight with 25 U/ml IL-2 and monocyte-depleted PBMCs (50:1). (A,B) Necrosis induction in JAK1-deficient and Sc hTERT immortalized urothelial cell lines. One-way ANOVA with Dunn's multiple comparisons post-test. (C) Relative change of necrosis compared to untreated. Two-tailed Mann Whitney U -test. Data is from five independent experiments. * P < 0.05. Error bars represent the SE.

Article Snippet: Mutations and their effects were presented against the JAK1 protein sequence (InterPro P23458).

Techniques: Cell Culture, Two Tailed Test, MANN-WHITNEY