Journal: BMC Pulmonary Medicine

Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

doi: 10.1186/s12890-021-01553-z

Figure Lengend Snippet: EML4-ALK transfection activated JAK2, STAT1, STAT3, STAT5, and STAT6 in HEK293 and NIH3T3 cells

Article Snippet: For immunohistochemistry, the sections were then incubated with the primary antibody against ALK (1:50, DAKO North America, USA), phosphorylated JAK2 (p-JAK2), phosphorylated STAT3 (P-STAT3), or phosphorylated STAT6 (p-STAT6) (1:50, Cell Signaling Technology, Inc., USA) at 4 °C overnight.

Techniques: Transfection

Journal: BMC Pulmonary Medicine

Article Title: EML4-ALK-mediated activation of the JAK2-STAT pathway is critical for non-small cell lung cancer transformation

doi: 10.1186/s12890-021-01553-z

Figure Lengend Snippet: IL4 and IL6 affected the JAK2-STAT pathway in EML4-ALK-positive cells. a STAT3 and STAT6 were phosphorylated and translocated to the nuclei of H2228 cells following IL4 or IL6 treatment. b Microarray analysis of H2228 cells stimulated or not with IL4. The heat map identifies modified proteins associated with the JAK2-STAT pathway, the regulation of the actin cytoskeleton, the cell cycle, cell adhesion, and the positive regulation of cell proliferation

Article Snippet: For immunohistochemistry, the sections were then incubated with the primary antibody against ALK (1:50, DAKO North America, USA), phosphorylated JAK2 (p-JAK2), phosphorylated STAT3 (P-STAT3), or phosphorylated STAT6 (p-STAT6) (1:50, Cell Signaling Technology, Inc., USA) at 4 °C overnight.

Techniques: Microarray, Modification

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 1. Ablation of Stat3 in bone marrow cells gives rise to skewed myeloid cell differentiation and hypersensitivity to GM-CSF. A, morphological, histological, and flow cytometric analyses of spleens from Stat3f/f/btie2-cre and Stat3f/f littermate control mice. Panel a, splenomagaly was found in Stat3f/f/ btie2-cre mice; panel b, comparison of spleen weight to body weight ratio of Stat3f/f and Stat3f/f/btie2-cre mice, p 0.01 (Student’s t test, 2 tails); panels c and d, disrupted spleen architecture in Stat3f/f/btie2-cre mice (d) when compared with Stat3f/f controls (c); panel e, both F480Mac1 and GR-1Mac1 popula- tions were significantly increased in Stat3f/f/btie2-cre spleens compared with the Stat3f/f control spleens. B, bone marrow F480Mac1 and GR-1Mac1 populations were increased in the Stat3f/f/btie2-cre mice compared with Stat3f/f control mice. C, methylcellulose-based colony forming assays demonstrated increased bone marrow progenitor-derived colonies in response to increasing doses of GM-CSF from the Stat3f/f/btie2-cre mice compared with the Stat3f/f mice. D,increasedproliferativeactivityofStat3f/f/btie2-crebonemarrowcellsinresponsetovariouscytokines.Thefinalconcentrationofallcytokines,exceptIL-3(100u/ml), was 30 ng/ml. E, decreased apoptosis in Stat3f/f/btie2-cre bone marrow cells when cultured in a low serum in the presence of 0.1 or 1 ng/ml of GM-CSF. Error bars represent S.D.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Cell Differentiation, Control, Comparison, Derivative Assay, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 2. Shp2 gain-of-function mutants negatively regulate Stat3 acti- vationinmurinebonemarrowcellsandperipheralbloodnucleatedcells from individuals with NS. A, panel a, Western blot analyses examining GM- CSF-stimulated (20 ng/ml) Stat3 activation in the mouse bone marrow-de- rived macrophage progenitors transduced with MIEG3 (empty vector), WT Shp2, or Shp2E76K. Panel b, density quantification of protein band in A, panel a; B, panel a, representative flow cytometric analysis showing reduced Stat3 tyrosine phosphorylation in peripheral blood cells from individuals with NS in both the quiescent state and after IL-6 stimulation. Panel b, statistical analysis

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Western Blot, Activation Assay, Transduction, Plasmid Preparation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 3. Stat3activationinvalvulogenesis,andShp2gain-of-functionmutantsinhibittheactivationofStat3inresponsetoEGF.A,immunohistochemistry staining of activated Stat3 (pY Stat3) in developing and mature PV of wild type mice. Nuclear brown signals are positive staining. Activated Stat3 is apparent in E14.5 embryonic heart and is restricted to the developing leaflets (A, panels a to c), and remains into adult stage (panel d). Activated Stat3 is present in the PV of control embryo hearts at E17.5 (panel e), absent in PV from Stat3f/f/nestin-cre embryo heart at the same stage (panel f). B, panel a, after serum deprivation overnight, Western blotanalysiswasusedtoexaminebasalandEGF-stimulated(50ng/ml)Stat3tyrosinephosphorylationinNIH3T3cellsexpressingShp2mutants(N308DandE76K)or controls(MIEG3orWTShp2).Panelb,densityquantificationoftheproteinbandinB,panela.C,EMSAtoexamineStat3DNAbindingactivityusingthem67-SIEprobe and nuclear extracts from NIH3T3 cells transduced with MIEG3 (M), WT Shp2 (WT), Shp2N308D (N308D), or Shp2E76K (E76K). Error bars represent S.D.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Staining, Control, Western Blot, Transduction

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 4. Conditional deletion of Stat3 with nestin-cre induces pulmonary stenosis due to the thicken- ing of leaflets. A, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside(X-gal) staining of nestin-cre/Rosa26R mouse embryos and newborns indicates the cre activity in the developing mouse embryos. A, panels a and b, central nervous system is positive for cre activity in the mouse embryos at E10.5. Panels c and d, cre activity was found in the pulmonary valves (PV) and the aortic valves (AV) at E14.5 (A, panel c) and P0 (B, panel d). B, ablation of Stat3 with nestin-cre leads to pulmonary stenosis caused by the thickening and enlargement of leaflets. B, panel a, newborn of Stat3f/f (f/f) and Stat3f/f/Nestin-cre (c;f/f) mice. B, panel b, hematoxylin and eosin staining of the transverse section of heart from control (f/f) and STAT3f/f/Nestin-cre (c;f/f) newborn mice (P0). Note that Stat3 ablation leads to right ventricle dilation in Stat3f/f/Nestin-cre mice. B, panels d–g, hematoxylin and eosin staining of sections of the pulmonary valve and aorta valve regions in control (B, panels d and f) and Stat3f/f/ Nestin-cre mutant (B, panels e and g) newborn mice. LV, left ventricle; RV, right ventricle. B, panel h, quantifica- tionofpulmonaryvalvethicknessinStat3f/f/Nestin-creandcontrolhearts(P0)(n6,p0.03,Student’spaired t test; 2 tails).

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Staining, Activity Assay, Control, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 5. Shp2 dephosphorylates Tyr(P)-Stat3 and Shp2-Stat3-medi- atedsignalingcriticallycontributestothepathogenesisofNS.A,Western blot analysis of Stat3-deficient bone marrow-derived macrophage progeni- tors treated with GM-CSF to assess Stat3 and ERK activation. B, panel a, puri- fied constitutively active Shp2 protein (catalytic domain) was capable of dephosphorylating Tyr(P)-Stat3. Stat3 protein was immunoprecipitated from IL-6-treated Raw 264.7 cell extract, and then incubated with purified recom- binant Shp2 protein (active form, 2 g) for the indicated time with or without preincubation of the small molecule Shp2 phosphatase inhibitor IIB-08 (100 M). Panel b, Shp2 inhibitor IIB-08 inhibits ERK activation, whereas enhancing the Tyr(P)-Stat3 level, in IL-6 induced Raw 264.7 cells. Raw 264.7 cells were pretreated with the Shp2 inhibitor IIB-08 (10 M), or dimethyl sulfoxide (DMSO) for 60 min, and then incubated with IL-6 (20 ng/ml) for 60 min before being subjected to lysis and Western blot analysis.

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Western Blot, Derivative Assay, Activation Assay, Immunoprecipitation, Incubation, Purification, Lysis

Journal: Journal of Biological Chemistry

Article Title: Negative Regulation of Stat3 by Activating PTPN11 Mutants Contributes to the Pathogenesis of Noonan Syndrome and Juvenile Myelomonocytic Leukemia

doi: 10.1074/jbc.m109.020495

Figure Lengend Snippet: FIGURE 7. Schematic diagram of dual-signaling pathways regulated by Shp2. In normal physiological conditions, Stat3 activity is under the positive and negative regulation of Janus kinases and Shp2, respectively (A). Shp2 gain-of-function mutations lead to the hyperactivation of the RAS/ERK signaling pathway and the excessive inactivation of Stat3, which synergistically promotes the pathogenesis of Noonan syndrome and/or JMML (B).

Article Snippet: To analyze phospho-Stat3 expression in the developing heart valve, we used a rabbit monoclonal antibody against phosphor-Stat3 (Tyr705) (Cell Signaling, D3A7) and a Vector staining system (Vector, PK-2200) according to the manufacturer’s instructions.

Techniques: Protein-Protein interactions, Activity Assay

Journal: Biochemical pharmacology

Article Title: Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis.

doi: 10.1016/j.bcp.2023.115523

Figure Lengend Snippet: Fig. 6. Lyn knockout aggravates kidney injury by activating phosphorylation of STAT3. Wild-type mice (Lyn+/+) or mice lacking the Lyn gene (Lyn−/−) were subjected to sham surgery or CLP (A-B). Some mice were also pretreated with the STAT3 inhibitor Stattic at 3 h before CLP (C-G). (A) Immunohistochemical staining of p-STAT3 in the kidney tissues. (B) Immu noblotting of p-STAT3 protein expression and density analysis of p-STAT3 expression in the kidney tissues. (C) Representative images of H&E and PAS staining of kidney tissue sections (magnification, 400×; bar = 50 μm). (D) Tubular injury scores of H&E staining for kidney damage. (E) BUN and sCr levels. (F) IL-1β, IL- 6, and TNF-α concentrations in mice serum of different groups. (G) Western blot and density anal ysis of p-STAT3, Bcl-2, Cleaved Caspase-3 and Bax in the kidney tissues. Data are representative of three independent experiments. Bars are represented as mean ± SD (n = 5–6 per group, one-way ANOVA with Tukey-Kramer post-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Next, slices were blocked with 3% BSA at room temperature for 30 min and incubated with anti-F4/80 (Cat# ab90247, RRID:AB_10712189; Abcam, Cambridge, MA, USA), anti-phosphorylated Lyn (Cat# 2731, RRID:AB_2138262; Cell Signaling Technology, Danvers, MA, USA) and anti-phosphorylated STAT3 (Cat# 9145, RRID:AB_2491009; Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C overnight.

Techniques: Knock-Out, Phospho-proteomics, Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Biochemical pharmacology

Article Title: Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis.

doi: 10.1016/j.bcp.2023.115523

Figure Lengend Snippet: Fig. 7. Lyn reduces apoptosis, inflammation and phosphorylation of STAT3 in LPS-treated BUMPT cells. (A) Western blot analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3. Cells were transfected with Lyn siRNA (50 nM) or negative control for 24 h before stimulation with 100ug/ml LPS for 4 h (p-STAT3, STAT3) and 24 h (Bcl-2, Cleaved Caspase-3). (B) Density analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3 (siRNA transfection). (C) The mRNA levels of inflammatory markers TNF-α, IL-6 and IL-1β in cell supernatant. Cells were transfected with Lyn siRNA (50 nM) or negative control for 24 h before stimula tion with 100ug/ml LPS for 24 h. (D) Western blot analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3. Cells were transfected with Lyn plasmid or negative control for 24 h before stimulation with 100ug/ml LPS for 8 h (p-STAT3, STAT3) and 24 h (Bcl-2, Cleaved Caspase-3). (E) Density analysis of p-STAT3, Bcl-2 and Cleaved Caspase-3 (Lyn plasmid trans fection). Data are representative of three independent experiments. Bars are represented as mean ± SD (n = 3 per group, one-way ANOVA with Tukey-Kramer post-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Next, slices were blocked with 3% BSA at room temperature for 30 min and incubated with anti-F4/80 (Cat# ab90247, RRID:AB_10712189; Abcam, Cambridge, MA, USA), anti-phosphorylated Lyn (Cat# 2731, RRID:AB_2138262; Cell Signaling Technology, Danvers, MA, USA) and anti-phosphorylated STAT3 (Cat# 9145, RRID:AB_2491009; Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C overnight.

Techniques: Phospho-proteomics, Western Blot, Transfection, Negative Control, Plasmid Preparation

Journal: Biochemical pharmacology

Article Title: Lyn attenuates sepsis-associated acute kidney injury by inhibition of phospho-STAT3 and apoptosis.

doi: 10.1016/j.bcp.2023.115523

Figure Lengend Snippet: Fig. 8. Lyn agonist MLR-1023 protects CLP-induced AKI by inhibiting STAT3 phosphorylation. (A) BUN and sCr levels. Wild-type mice were pretreated with MLR-1023 (10, 30, 50 mg/kg i.p.) or DMSO 2 h before sham surgery or CLP. After 16 h, blood samples were collected. (B) Representative images of H&E and PAS staining of kidney tissue sections (Magnification: 400x, scale bar: 50 μm). Wild-type mice were pre treated with MLR-1023 (50 mg/kg i.p.) or DMSO 2 h before sham surgery or CLP. After 16 h, kidney tissues and blood samples were collected. (C) Renal tubular injury score. (D) BUN and sCr levels. (E) Elisa assay of serum IL-1β, IL-6, and TNF-α. (F) Western blot anal ysis of p-STAT3, Bcl-2 and Cleaved Caspase-3 in renal tissues. (G) Density analysis of p-STAT3, p-Lyn, Bcl-2 and Cleaved Caspase-3. Data are representative of three independent experiments. Bars are represented as mean ± SD (n = 5–6 per group, one-way ANOVA with Tukey-Kramer post-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Next, slices were blocked with 3% BSA at room temperature for 30 min and incubated with anti-F4/80 (Cat# ab90247, RRID:AB_10712189; Abcam, Cambridge, MA, USA), anti-phosphorylated Lyn (Cat# 2731, RRID:AB_2138262; Cell Signaling Technology, Danvers, MA, USA) and anti-phosphorylated STAT3 (Cat# 9145, RRID:AB_2491009; Cell Signaling Technology, Danvers, MA, USA) at 4 ◦C overnight.

Techniques: Phospho-proteomics, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: OMICS : a Journal of Integrative Biology

Article Title: Chronic Cigarette Smoke Mediated Global Changes in Lung Mucoepidermoid Cells: A Phosphoproteomic Analysis

doi: 10.1089/omi.2017.0090

Figure Lengend Snippet: Chronic exposure to cigarette smoke increases invasive potential of cells and enhances cell survival proteins. (A) Cellular morphology of parental (H292-P) and smoke treated cells (H292-S). (B) Invasive ability of parental (H292-P) and smoke treated cells (H292-S) (*p < 0.05). (C) Western blot analysis of cell survival proteins in H292-P and H292-S cells (D) Western blot analysis of epithelial-mesenchymal transition marker proteins in H292-P and H292-S. β-actin serves as a loading control. (E) Western blot analysis of STAT3 (S727) and NF-κB (S276 and S468) activation sites in H292-P and H292-S. NF-κB, NF-kappa-B; STAT, signal transducer and activator of transcription.

Article Snippet: The antibodies against following molecules were purchased from Cell Signaling Technology—Bcl-xL, Bcl-2, Bax, E-Cad, N-Cad, Slug, Snail, Twist, STAT3, phosphor-STAT3 (S727), NF-κB, phospho-NF-κB (S276 and S468), AKT, and phospho-AKT (S473 and T308).

Techniques: Western Blot, Marker, Activation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus

doi: 10.1016/j.jcmgh.2023.04.006

Figure Lengend Snippet: TMEM164 and STAT3 activation/ IL6 expression in TMNK1 cells. ( A ) Suppression of TMEM164 expression by TMEM164 siRNA. ∗∗∗∗ P < .0001, vs control subjects. ( B ) Western blotting analysis of phosphorylated STAT3 expression in cells treated with TMEM164 siRNA. Suppression of TMEM164 expression resulted in a decrease of phosphorylated STAT3 levels. ( C ) Enzyme-linked immunosorbent assay measuring IL6 in cells treated with TMEM164 siRNA. Suppression of TMEM164 expression resulted in a decrease of IL6 levels. ∗∗∗∗ P < .0001, vs control subjects. ( D ) Vectors expressing different TMEM164 variants (Vt1 and Vt2). ∗∗∗∗ P < .0001, vs control subjects. ( E ) Western blotting analysis of phosphorylated STAT3 levels in cells overexpressing Vt1 or Vt2. Overexpression of Vt1 or Vt2 resulted in an increase of phosphorylated STAT3 levels. ( F ) Enzyme-linked immunosorbent assay measuring IL6 in cells overexpressing Vt1 or Vt2. Overexpression of Vt1 or Vt2 resulted in an increase of IL6 levels.

Article Snippet: Protein expression analysis in liver tissue included primary antibodies targeting ATF6 (Abcam), IL6 (Abcam), total STAT3 (Cell Signaling Technology), phosphorylated-STAT3 (Tyr705) (Cell Signaling Technology), and GAPDH (Santacruz).

Techniques: Activation Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus

doi: 10.1016/j.jcmgh.2023.04.006

Figure Lengend Snippet: Association of TMEM164 with ATF6 and GRP78/BIP. ( A ) Western blotting analysis of the expression of full-length ATF6 and cleaved ATF6 when TMEM164 was overexpressed or suppressed. Full-length ATF6 and cleaved ATF6 were expressed in TMNK1 cells overexpressing TMEM164 . Knockdown of TMEM164 by siRNA resulted in a decrease of full-length ATF6 expression. ( B ) Immunoprecipitation examining binding of TMEM164 to full-length ATF6 and GRP78/BIP using cells overexpressing TMEM164 Vt1 or Vt2. Both variants bound to full-length ATF6 and GRP78/BIP. ( C ) Analysis of ATF6 , IL6 , and TMEM164 expression in cells overexpressing TMEM164 Vt1 or Vt2, with or without ATF6 siRNA. ATF6 and IL6 were significantly upregulated by TMEM164 Vt1 or Vt2 overexpression. ATF6 siRNA suppressed ATF6 expression and decreased IL6 levels (left and center). In contrast, suppression of ATF6 expression resulted in a decrease of TMEM164 expression (right). ∗∗ P < .01, ∗∗∗ P < .001, ∗∗∗∗ P < .0001, vs control subjects. ( D ) Enzyme-linked immunosorbent assay measuring IL6 in cells overexpressing TMEM164 Vt1 or Vt2, with or without ATF6 siRNA. ( C ) Changes in IL6 protein levels were similar to those at the mRNA level (center). ∗∗∗ P < .001, ∗∗∗∗ P < .0001, vs control subjects. ( E ) Western blotting analysis of total STAT3 and p-STAT3 in cells overexpressing TMEM164 Vt1 or Vt2, with or without ATF6 siRNA. Overexpression of TMEM164 Vt1 and Vt2 increased p-STAT3 levels, but this was restored to background levels by the suppression of ATF6 expression. ( F ) Illustration depicting the relationship between TMEM164, ATF6, and BIP in the ER lumen. TMEM164 binds to GRP78/BIP, increases the levels of cleaved ATF6 and full-length ATF6, and is involved in increases in the levels of IL6 and p-STAT3. TMEM164 could be regulated by ATF6 epigenetically and transcriptionally.

Article Snippet: Protein expression analysis in liver tissue included primary antibodies targeting ATF6 (Abcam), IL6 (Abcam), total STAT3 (Cell Signaling Technology), phosphorylated-STAT3 (Tyr705) (Cell Signaling Technology), and GAPDH (Santacruz).

Techniques: Western Blot, Expressing, Immunoprecipitation, Binding Assay, Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus

doi: 10.1016/j.jcmgh.2023.04.006

Figure Lengend Snippet: TMEM164 KO reduces ATF6-mediated endoplasmic reticulum stress and liver inflammation in a carbon tetrachloride–induced liver fibrosis mouse model. ( A ) Liver tissue slides of TMEM164 KO mice and control mice after exposure to carbon tetrachloride for 4 weeks were stained with 8-hydroxy-2′-deoxyguanosine (8-OHdG). ( B ) Analysis of inflammatory cytokines in liver tissues of TMEM164 KO mice. The expression of IL1b, tumor necrosis factor (TNF), and IL6 was significantly decreased. ∗ P < .05, ∗∗ P < .01, vs WT control subjects. ( C ) Western blotting of ATF6, IL6, STAT3, p-STAT3, TMEM164, and GAPDH in liver tissues from TMEM164 KO and control mice after exposure to carbon tetrachloride for 4 weeks. ( D ) Lx-2 human hepatic stellate cells were transfected using stealth siRNA Human TMEM164 with Lipofectamine RNAiMAX. After 48 hours, RNA was extracted and ACTA2 , COL1A1 , COL1A2 , and TMEM164 expression was analyzed by real-time PCR using TaqMan probes.

Article Snippet: Protein expression analysis in liver tissue included primary antibodies targeting ATF6 (Abcam), IL6 (Abcam), total STAT3 (Cell Signaling Technology), phosphorylated-STAT3 (Tyr705) (Cell Signaling Technology), and GAPDH (Santacruz).

Techniques: Staining, Expressing, Western Blot, Transfection, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Identification of a Transmembrane Protein Involved in Shear Stress Signaling and Hepatocarcinogenesis After a Sustained Virological Response to Hepatitis C Virus

doi: 10.1016/j.jcmgh.2023.04.006

Figure Lengend Snippet: Isolation of nonparenchymal cells from xenograft tumors of Huh7 or KH cells with TMNK1 cells overexpressing TMEM164 and the evaluation of the gene expression of human angiogenic factors. ( A ) Nonparenchymal cells were isolated from xenograft tumors of Huh7 cells with TMNK1 cells overexpressing TMEM164 Vt1 or Vt2 by Percoll gradient centrifugation. The expression of human EGF , TGFB1 , VEGFA , and WNT5A was analyzed by real-time PCR using TaqMan probes. ( B ) Nonparenchymal cells were isolated from xenograft tumors of KH cells with TMNK1 cells overexpressing TMEM164 Vt1 or Vt2 by Percoll gradient centrifugation. The expression of human EGF , TGFB1 , and VEGFA was analyzed by real-time PCR using TaqMan probes. ( C ) Immunohistochemical staining of phosphorylated signal transducer and activator of transcription 3 (STAT3) in xenograft tumors of Huh7 or KH cells with TMNK1 cells overexpressing TMEM164 Vt1 or Vt2.

Article Snippet: Protein expression analysis in liver tissue included primary antibodies targeting ATF6 (Abcam), IL6 (Abcam), total STAT3 (Cell Signaling Technology), phosphorylated-STAT3 (Tyr705) (Cell Signaling Technology), and GAPDH (Santacruz).

Techniques: Isolation, Expressing, Gradient Centrifugation, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Loss of Erbin induces STAT3 activation in cervical cancer cells. ( a , b ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% or 10% or no serum. The phosphorylations of FAK ( a ) and Src ( b ) were analyzed by western blot. ( c ) Parental HeLa (HeLa/WT), HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% or no serum. The phosphorylation of STAT3 was analyzed by western blot. The relative STAT3 phosphorylation levels were determined by densitometry and normalized with protein levels. ( d ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% serum in the plates coated with PolyHEMA. The phosphorylation of STAT3 was analyzed at the indicated time points. The relative STAT3 phosphorylation levels were determined by densitometry and normalized with protein levels. ( e ) HeLa/NC and HeLa/Erbin-sh cells were cultured in serum-free media. The phosphorylation of STAT3 was analyzed at the indicated time points. ( f ) HeLa/NC and HeLa/Erbin-sh cells were cultured in serum-free media overnight and then treated with 10 μℳ ISO. The phosphorylation of STAT3 was analyzed at the indicated time points. ** P <0.01.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Activation Assay, Cell Culture, Western Blot, Phospho-proteomics

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Loss of Erbin expression confers resistance of cervical cancer cells to anoikis in a STAT3-dependent manner. ( a , b ) HeLa cells were transfected with the plasmid expressing STAT3C (HeLa/STAT3C) or the empty vector (HeLa/Vector). The transfected cells were cultured in the medium containing 1% serum in the plates coated with PolyHEMA. After incubation for 48 and 72 h, the anoikis rates were determined by FACS. ( c , d ) HeLa/NC and HeLa/Erbin-sh cells cultured in suspension were treated with WP1066. The anoikis rates were determined by FACS. ( e , f ) HeLa/NC and HeLa/Erbin-sh cells under conventional culture were treated with WP1066. The apoptotic rates were determined by FACS. ( g , h ) HeLa/NC and HeLa/Erbin-sh cells were treated with WP1066, U0126 or PD98059. The anoikis rates were determined by FACS. ** P <0.01. DMSO, dimethyl sulfoxide; PI, propidium iodide.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, Incubation, Suspension

Journal: Oncogenesis

Article Title: Deficiency of Erbin induces resistance of cervical cancer cells to anoikis in a STAT3-dependent manner

doi: 10.1038/oncsis.2013.18

Figure Lengend Snippet: Erbin negatively regulates IL-6/STAT3 pathway. ( a ) HeLa/NC and HeLa/Erbin-sh cells under conventional culture were treated with 10 ng/ml IL-6. The activation of STAT3 was analyzed by western blot at the indicated time points. ( b ) HeLa/NC and HeLa/Erbin-sh cells were treated with 10 ng/ml IL-6 and then labeled with the anti-phoaphor-STAT3 antibody and Alexa fluor 549-labeled secondary antibody. Nuclei were stained with 1 μg/ml DAPI. Nuclear translocation of activated STAT3 was observed under a laser scanning confocal microscope. ( c ) HeLa/NC and HeLa/Erbin-sh cells grown in 24-well plates were transiently cotransfected by the STAT3 reporter and pRL-TK vectors. The transfected cells were treated with or without 10 ng/ml IL-6. The luciferase activities were measured using a dual luciferase assay kit. ( d , e ) HeLa/NC and HeLa/Erbin-sh cells were cultured in the media containing 1% serum in the plates coated with PolyHEMA for 24 h and treated with or without 10 ng/ml IL-6. The phosphorylation of STAT3 was analyzed ( d ) and the luciferase activities were measured ( e ). ( f ) HeLa cells were treated with or without IL-6, and the expression of Erbin mRNA was analyzed by real-time reverse transcriptase–PCR (RT-PCR). ( g ) HeLa cells were transfected with pRc/CMV-Stat3C-Flag (HeLa/STAT3C) or the empty vector (HeLa/Vector). The expression of Erbin mRNA was examined by real-time RT-PCR. ( h ) The HeLa/STAT3C and HeLa/Vector cells were transfected with Erbin siRNA or control siRNA. The expression of Erbin protein was examined by western blot. ( i ) HeLa cells were starved overnight and then treated with AG490. IL-6 was added into the cell culture after 1 h of the treatment. The phosphorylation of STAT3 was analyzed by western blot at the indicated time points. ** P <0.01.

Article Snippet: The HeLa cells were treated with 10 ng/ml IL-6 for different time periods and then labeled with the rabbit monoclonal antibody against phosphor-STAT3 (Tyr705, Cell Signaling).

Techniques: Activation Assay, Western Blot, Labeling, Staining, Translocation Assay, Microscopy, Transfection, Luciferase, Cell Culture, Phospho-proteomics, Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Quantitative RT-PCR, Control