Journal: International journal of oncology

Article Title: B-cell CLL/lymphoma 3 promotes glioma cell proliferation and inhibits apoptosis through the oncogenic STAT3 pathway.

doi: 10.3892/ijo.2016.3729

Figure Lengend Snippet: Figure 8. STAT3 knockdown reverses the oncogenic role of BCL3 in glioma cells. (A) Western blot analyses confirmed that STAT3 expression was knocked down by siRNA in BCL3 overexpressing U87 cells. n=3. (B) STAT3 knockdown evidently reduced proliferation in BCL3 overexpressing U87 cells. n=3, *P<0.05. (C) The proliferative ability of BCL3 overexpressing U87 cells were prominently reduced after STAT3 knockdown. n=3, *P<0.05. (D) STAT3 knockdown led to G1-phase arrest in BCL3 overexpressing U87 cells. n=3, *P<0.05. (E) STAT3 knockdown resulted in an obviously increased percentage of apoptotic cells in BCL3 overexpressing U87 cells. n=3, *P<0.05.

Article Snippet: A STAT3 specific siRNA and scrambled siRNA was obtained from OriGene Technologies, Inc. (Shanghai, China).

Techniques: Knockdown, Western Blot, Expressing

Journal: Frontiers in Oncology

Article Title: RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells

doi: 10.3389/fonc.2022.826617

Figure Lengend Snippet: STAT3 is not a primary target altering cellular sensitivity to Ref-1 inhibitors in murine PDAC Kras G12D cells. (A) STAT3 was knocked down in KC3590 cells that are RelA deficient (Vector) and clone with functional RelA add back (C13) and knockdown efficiency was assessed by Western bot. Vinculin was used as loading control. The cells were challenged for 48 hours with (B) APX3330, (C) APX2009, (D) APX2014, and (E) RN7-58. Cytotoxicity was measured by alamarBlue. At least three independent experiments were performed (N = 3).

Article Snippet: Cells were transfected by lipofectamine RNAiMax (Invitrogen, CA, USA) with PRDX1 (SR405074, OriGene Technologies, MD, USA), RelA (SR417160, OriGene Technologies, MD, USA), STAT3 (SR427487, OriGene Technologies, MD, USA), and universal scrambled control (SCR) (SR30004, OriGene Technologies, MD, USA) siRNAs.

Techniques: Plasmid Preparation, Functional Assay, Knockdown, Western Blot, Control

Journal: Journal of Cell Science

Article Title: RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

doi: 10.1242/jcs.209932

Figure Lengend Snippet: Validating screen hits in IPF patient fibroblasts. Fifty-three selected hits from gel plate screening were tested in three IPF-derived primary human lung fibroblast cultures grown on gel plates, with αSMA fibers and intracellular procollagen immunostaining quantified. Confirmation by this approach required at least one siRNA per target gene to score positively in at least two cell lines and consistently in two replicate experiments. (A) 37 genes (of 53 tested) were validated by αSMA fiber assay. (B) 21 of these 37 validated genes also affected cellular procollagen levels. Percentage change from non-targeting control siRNA is shown. Averages are taken from three IPF cell lines and represent the single siRNA duplex with the strongest effect. Error bars represent s.d. (C) Plot of IMR-90 gel plate and average IPF fibroblast αSMA assay data for 53 gene targets tested. (D) Representative images of three IPF fibroblast cultures transfected with non-targeting control or STAT3 siRNAs. αSMA fiber or procollagen signal normalized to same plate non-targeting control siRNA is shown. A zoomed-in view is presented to show detail but quantification is from a full field of view. Scale bars: 20 µm.

Article Snippet: STAT3 ELISA NHLF cells (2×10 5 cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS.

Techniques: Derivative Assay, Immunostaining, Control, Transfection

Journal: Journal of Cell Science

Article Title: RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

doi: 10.1242/jcs.209932

Figure Lengend Snippet: STAT3 is essential for myofibroblast pro-fibrotic gene expression, contractility and matrix deposition. (A) Confirmation of STAT3 knockdown by siRNA. Experiment was performed in triplicate in IMR-90 fibroblasts transfected with non-targeting (NT) or STAT3 siRNA in the presence of TGF-β. Error bars represent s.e.m. (*P<0.05 vs non-targeting control siRNA). (B) RNA from IMR-90 cells transfected with NT or STAT3 siRNAs in the presence or absence (Control) of TGF-β (2 ng/ml, 72 h) was isolated to determine expression of pro-fibrotic genes ACTA2, COL1A2 and CTGF relative to GAPDH. Experiment was performed in triplicate and error bars represent s.e.m. (*P<0.05, **P<0.01, ****P<0.0001 vs non-targeting control siRNA). (C) STAT3 expression regulates myofibroblast contractility measured by traction force microscopy. Representative traction maps are shown from IMR-90 cells plated onto 6.4 kPa matrices transfected with non-targeting control or STAT3 siRNAs in the presence of TGF-β. RMS tractions were determined in two independent experiments; data shown are individual cell tractions and means (±s.e.m.) from one representative experiment (**P<0.01 vs non-targeting control siRNA). Scale bars: 20 µm. (D) Immuno-ECM assay to measure matrix deposition. IMR-90 cells were transfected with NT or STAT3 siRNA for 48 h and plated at confluence for an additional 48 h in the presence of TGF-β. Cell-derived matrices were then probed for fibronectin and collagen I. Experiment was performed in triplicate and error bars represent s.e.m. (*P<0.05, **P<0.01 vs non-targeting control siRNA).

Article Snippet: STAT3 ELISA NHLF cells (2×10 5 cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS.

Techniques: Gene Expression, Knockdown, Transfection, Control, Isolation, Expressing, Microscopy, Derivative Assay

Journal: Journal of Cell Science

Article Title: RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

doi: 10.1242/jcs.209932

Figure Lengend Snippet: Essential role of STAT3 role in myofibroblast activation is conserved across multiple organs. The small-molecule inhibitor of STAT3, LLL12, was used to pharmacologically inhibit STAT3. (A) LLL12 reduces expression of αSMA. IMR90 cells were stimulated with 2 ng/ml TGF-β for 72 h ±1 µM LLL12 for the final 24 h then immunostained for αSMA and DAPI. Shown are representative images. The percentage of αSMA-positive cells was determined in a blinded fashion in three replicate experiments using an optical threshold, with the mean across these experiments indicated in the upper corner of each image. Scale bars: 20 µm. (B) LLL12 reduces matrix protein deposition. IMR-90 cells were plated to confluence and allowed to deposit matrix for 72 h ±2 ng/ml TGF-β and ±LLL12. Cells were then lysed leaving cell-derived matrix which was immunostained for fibronectin or collagen I and quantified using infrared secondary antibodies. Representative immunostained images are shown and plots indicate the mean (±s.e.m.) from three independent experiments normalized to DMSO (*P<0.05, ****P<0.0001 vs TGF-β+DMSO). (C) LLL12 reduces pro-fibrotic gene expression. IMR90 cells were stimulated with 2 ng/ml TGF-β for 72 h ±LLL12 for the final 24 h prior to RNA isolation. Expression of ACTA2, COL1A2, FN1 and CTGF was measured relative to GAPDH. Data shown are the mean (±s.e.m.) from three independent experiments normalized to DMSO (**P<0.01, ***P<0.001, ****P<0.0001 vs DMSO). (D) LLL12 reduces cellular contractility. IMR-90 were plated onto 6.4 kPa matrices for traction force microscopy in the presence of TGF-β, ±LLL12 or ±20 µM Y-27632 (ROCK inhibitor). RMS traction forces were determined from two independent experiments and shown are individual cell tractions and means (±s.e.m.) from one representative experiment (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO). (E) LLL12 reduces pro-fibrotic gene expression in primary human hepatic stellate cells, primary human cardiac fibroblasts and IPF patient-derived lung fibroblasts. Hepatic stellate cells and cardiac fibroblasts were treated with TGF-β for 72 h, IPF fibroblasts were cultured in control medium for the same time. All cells were treated with 0.3 µM LLL12 for the final 24 h; data show the mean (±s.e.m.) from three independent experiments normalized to DMSO-treated control for each cell type (*P<0.05, **P<0.01, ***P<0.001 vs DMSO). (F) LLL12 reduces cellular contractility in primary human hepatic stellate cells, primary human cardiac fibroblasts and IPF patient-derived lung fibroblasts. Cells were treated as in E; shown are individual cell tractions and mean (±s.e.m.) from one representative experiment (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs DMSO).

Article Snippet: STAT3 ELISA NHLF cells (2×10 5 cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS.

Techniques: Activation Assay, Expressing, Derivative Assay, Gene Expression, Isolation, Microscopy, Cell Culture, Control

Journal: Journal of Cell Science

Article Title: RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

doi: 10.1242/jcs.209932

Figure Lengend Snippet: STAT3 is constitutively activated on a stiff matrix independent of extracellular autocrine signaling. (A) IMR-90 lung fibroblasts were plated onto PA hydrogels with Young's modulus of 1 (soft) and 75 kPa (stiff) for 24 h prior to protein isolation and western blotting for total and phospho-STAT3 (Y705) (*P<0.05 vs soft). (B) Normal primary human lung fibroblasts were cultured on 1 kPa hydrogels or tissue culture plastic, and STAT3 phosphorylation (Y705) was assessed by ELISA and normalized to total protein (*P<0.05 vs soft). (C) IL-6 (50 ng/ml, 1 h) stimulation of lung fibroblasts increased STAT3 phosphorylation (Y705) to similar extents on plastic (stiff) and 1 kPa hydrogels (soft) (*P<0.05). (D) IMR-90 cells were plated onto soft PA hydrogels (1 kPa, Young's modulus) or tissue culture plastic for 24 h. Conditioned media from cells grown on plastic culture plates was transferred to the cells on 1 kPa hydrogels for 0.5, 1.0 and 2.0 h, with total and phospho-STAT3 (Y705) measured on western blots. (E,F) Lung fibroblasts were treated with (E) or without (F) IL-6 (50 ng/ml) after pre-treatment with neutralizing antibodies against IL-6r (30 µg/ml), gp130 (30 µg/ml) or control IgG (30 µg/ml), and STAT3 phosphorylation (Y705) was assessed by ELISA and normalized to total protein.

Article Snippet: STAT3 ELISA NHLF cells (2×10 5 cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS.

Techniques: Isolation, Western Blot, Cell Culture, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of Cell Science

Article Title: RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

doi: 10.1242/jcs.209932

Figure Lengend Snippet: Constitutive STAT3 phosphorylation on stiff matrices is dependent on ROCK and JAK2. Normal human lung fibroblasts were incubated with the indicated concentrations of ROCK inhibitor Y27632 or myosin II inhibitor Blebbistatin (A), JAK2 inhibitor AZD1408, JAK1/2 inhibitor Ruxolitinib or JAK3 inhibitor Tofacitinib (B), FAK inhibitor PF562271 or TGF-β type I receptor inhibitor SB431542 (C) for 1 h and STAT3 phosphorylation (Y705) was assessed by ELISA and normalized to total protein (*P<0.05, ***P<0.001, ****P<0.0001).

Article Snippet: STAT3 ELISA NHLF cells (2×10 5 cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS.

Techniques: Phospho-proteomics, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: RNAi screening identifies a mechanosensitive ROCK-JAK2-STAT3 network central to myofibroblast activation

doi: 10.1242/jcs.209932

Figure Lengend Snippet: STAT3 inhibition attenuates experimental lung fibrosis. (A) Representative hematoxylin and eosin (left)- and trichrome (right)-stained images from mice instilled intratracheally with saline (control) or bleomycin at day 0, then injected intraperitoneally with DMSO or LLL12 on days 14-28. Scale bars: 100 µm. (B) Trichrome-stained sections from each group were blindly scored for fibrosis using Ashcroft's scale. Data represent the mean (±s.e.m.) from each group (Control DMSO, N=10; Control LLL12, N=8; Bleomycin DMSO, N=6; Bleomycin LLL12, N=7) (****P<0.0001 vs Control DMSO; ####P<0.0001 vs Bleomycin DMSO). (C) Western blot analysis of STAT3 phosphorylation in total lung protein from each group. Data represent the mean (±s.e.m.) from each group, N=3 randomly selected from each group (#P<0.05 vs Bleo DMSO). (D) Lung collagen content was determined by measuring hydroxyproline content in total protein from each group. Data represent the mean (±s.e.m.) from each group, N=6 randomly selected from each group (****P<0.0001 vs Control DMSO; ##P<0.01 vs Bleomycin DMSO). (E) LLL12 reduces bleomycin induced pro-fibrotic gene expression. Expression of Acta2, Col1a2, Fn1 and Ctgf was measured relative to Gapdh. Data shown are the mean (±s.e.m.) from each group (N=6 randomly selected from each group) normalized to Control DMSO (*P<0.05, ***P<0.001, ****P<0.0001 vs Control DMSO; #P<0.05, ##P<0.01 vs Bleomycin DMSO).

Article Snippet: STAT3 ELISA NHLF cells (2×10 5 cells, Lonza) were plated onto soft hydrogel (1 kPa, Young's modulus) or tissue culture plastic in 12-well plates at 37°C for 24 h in FGM-2 medium (Lonza) containing 0.1% FBS.

Techniques: Inhibition, Staining, Saline, Control, Injection, Western Blot, Phospho-proteomics, Gene Expression, Expressing

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: Chemicals and reagents with their catalog numbers and companies.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Modification, Protein Concentration, Mutagenesis, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: Sequences of the primers for qPCR.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: Transfection plasmid combinations of dual luciferase.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Transfection, Plasmid Preparation, Luciferase

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: (A) predicted binding sites for miR-223-3P and STAT3, (B) changes in luciferase activity of added STAT3 3'-UTR pMIR-TM luciferase vector and miR-223-3p mimic, (C) STAT3 molecular level of added miR-223-3p mimic, (D) STAT3 protein level of added miR-223-3p mimic, (E) miR-223-3P and IL-6 predicted binding site, (F) changes in luciferase activity of added IL- 6 3'-UTR psi-CHECK2 luciferase vector and miR-223-3p mimic, (G) IL-6 molecular level of added miR-223-3p mimic, (H) IL-6 protein level of added miR-223 -3p mimic, (I) miR-2909 and KLF4 predicted binding sites, (J) changes in luciferase activity of added KLF4 3'-UTR pMIR GFP luciferase vector and miR-2909 mimic, (K) the molecular level of added KLF4 and miR-2909, (L) the protein level of added KLF4 and miR-2909. *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Binding Assay, Luciferase, Activity Assay, Plasmid Preparation

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: The molecular expression level and protein expression levels of IL-6, TNF-α, IL-1β, TLR4, TLR2, miR-223-3p and STAT3. (A-C) protein levels of IL-6, TNF-α, IL-1β, (D-J) molecular levels of IL-6, TNF-α, IL-1β, miR-223-3p, STAT3, TLR4, TLR2, (K) protein levels of TLR4, TLR2, STAT3, p-STAT3. *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: (A) STAT3 protein level after STAT3 siRNA transfection, (B-D) protein expression of IL-6, TNF-α, IL-1β after transfection of miR-223-3p and STAT3 siRNA, (E-I) molecular expression level of TLR2, TLR4, IL- 6. TNF-α and IL-1β, (J) protein expression of TLR2 and TLR4. *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Transfection, Expressing

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: (A-C) IL-6, TNF-α, IL-1β protein levels, (D-I) IL-6, TNF-α, IL-1β, STAT3, TLR4, TLR2 molecular expression levels, (J) TLR4, TLR2, STAT3, p-STAT3 protein expression levels. *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Impact of miR-223-3p and miR-2909 on inflammatory factors IL-6, IL-1ß, and TNF-α, and the TLR4/TLR2/NF-κB/STAT3 signaling pathway induced by lipopolysaccharide in human adipose stem cells

doi: 10.1371/journal.pone.0212063

Figure Lengend Snippet: (A-B) mir-223-3p and STAT3 molecular expression after transfection of IL-6, siIL-6, (C) STAT3 protein expression after transfection of siIL-6, (D) STAT3 and pSTAT3 protein expression after transfection of IL-6, siIL-6, (E-F) mir-223-3p and STAT3 molecular expression after transfection of TNF-α, siTNF-α, (G) STAT3 protein expression after transfection of siTNF-α, (H) STAT3 and pSTAT3 protein expression after transfection of TNF-α and siTNF-α. *** p <0.001; ** p <0.01; * p <0.05.

Article Snippet: STAT3 siRNA , , Ruibo Bio (Guangzhou, China).

Techniques: Expressing, Transfection