Journal: PLoS ONE

Article Title: Vaspin Increases Nitric Oxide Bioavailability through the Reduction of Asymmetric Dimethylarginine in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0052346

Figure Lengend Snippet: A–B. Effects of vaspin on STAT3 phosphorylation in HAECs according to the incubation time of vaspin (A) and dose of vaspin (B). HAECs were treated with 100 ng/ml of vaspin for the indicated time (A) and indicated concentrations of vaspin for 6 hr (B). C Effects of vaspin on STAT3-DNA binding activity. Electrophoretic mobility shift analysis (EMSA) was used to determine the DNA-binding activity of STAT3 in HAECs. Cells were treated with STAT3 probe or cold probe in the absence or the presence of 100 ng/ml of vaspin for 6 hr. Nuclear extracts from the treated and untreated control cells were isolated and used in an EMSA with biotin-labeled STAT3 oligonucleotide or cold probe. We used 2 ug and 4 ug of STAT3 Ab to identify the supershifts. The solid arrow and dashed arrow indicate the STAT3 binding complex and supershift, respectively. Data are representative results from three separate experiments. D. Effect of STAT3 siRNA on vaspin-induced eNOS phosphorylation in HAECs. HAECs were treated with control siRNA ± vaspin 100 ng/ml or STAT3 siRNA ± vaspin 100 ng/ml. Relative expression of STAT3 and eNOS were measured at 16 hr after vaspin treatment. Data are shown as means ± SEM of at least three independent experiments.In A: * p <0.05 vs. untreated (Control). In B: * p <0.05 vs. untreated (Control); # p <0.05 vs. 25 ng/ml of vaspin group. In D: * p <0.05 vs. control siRNA alone; # p <0.05 vs. control siRNA+vaspin. STAT3, signal transducer and activator of transcription 3; eNOS, endothelial nitric oxide synthase.

Article Snippet: Cells were transfected with 200 ng of STAT3-expression vector (pUNO1-hSTAT3b, InvivoGen, San Diego, CA, USA), or 200 ng of wild type or mutant type reporter gene using LipofectAMINE 2000 reagent (Invitrogen). pRL-SV40 renilla luciferase control reporter vectors (10 ng, Promega, Madison, WI, USA) were co-transfected as an internal control.

Techniques: Incubation, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Isolation, Labeling, Expressing

Journal: PLoS ONE

Article Title: Vaspin Increases Nitric Oxide Bioavailability through the Reduction of Asymmetric Dimethylarginine in Vascular Endothelial Cells

doi: 10.1371/journal.pone.0052346

Figure Lengend Snippet: The effect of STAT3-expression vector on STAT3 expression. The expressions of STAT3 at the level of protein (A) and mRNA were measured after 24 hr of transfection (B). C. The stimulatory effects of vaspin on STAT3-induced DDAH II expression. HAECs were transfected with 200 ng of STAT3-expression vector or control empty vector and then incubated with or without vaspin (100 ng/ml) for 16 hr. D. The role of STAT3-binding site in vaspin’s stimulatory effect on DDAH II expression. HAECs were transfected with DDAH II promoter-reporter plasmid (wild type or mutant type at putative STAT3-binding site) and stimulated with vehicle or vaspin (100 ng/ml) for 16 hr. Data are shown as means ± SEM of at least three independent experiments. In A and B: * p <0.05 vs. control vector. In C: * p <0.05 vs. untreated (Control); # p <0.05 vs. vaspin+control vector group; † p <0.05 vs. STAT3 vector alone group. In D: * p <0.05 vs. wild promoter only group; # p <0.05 vs. wild promoter+vaspin group. STAT3, signal transducer and activator of transcription 3; DDAH II, dimethylarginine dimethylaminohydrolase II.

Article Snippet: Cells were transfected with 200 ng of STAT3-expression vector (pUNO1-hSTAT3b, InvivoGen, San Diego, CA, USA), or 200 ng of wild type or mutant type reporter gene using LipofectAMINE 2000 reagent (Invitrogen). pRL-SV40 renilla luciferase control reporter vectors (10 ng, Promega, Madison, WI, USA) were co-transfected as an internal control.

Techniques: Expressing, Plasmid Preparation, Transfection, Incubation, Binding Assay, Mutagenesis

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Oligonucleotide primer sets for RT-qPCR.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Sequencing

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal transfer of NEAT1 to EC cells leads to increased expression of STAT3 and YKL-40. (A) RT-qPCR analysis of NEAT1 expression level in NFs, CAFs, normal endometrial epithelial cells, and EC cells. (B) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or exosome-depleted CAFs-CM for 48 h, then NEAT1 level was assessed by RT-qPCR. HEC-1A and RL95-2 cells were maintained in control CM, CAFs-CM, or 20 μM GW4869-treated CAFs-CM for 48 h. (C) NEAT1 expression was determined by RT-qPCR. (D) The NEAT1 level was assessed by RT-qPCR. (E) STAT3, p-STAT3 and YKL-40 levels were assessed by Western blot. HEC-1A and RL95-2 cells were co-cultured with CAFs or GW4869-treated CAFs for 48 h. (F) Western blot analysis of STAT3, p-STAT3, and YKL-40 levels. (G) Western blot analysis of Rab27a protein level in CAFs infected with lentivirus expressing si-NC or si-Rab27a. (H) HEC-1A and RL95-2 cells were cultured in control CM, CAFs-CM, or Rab27a-silenced CAFs-CM for 48 h, then NEAT1 level was detected by RT-qPCR. (I) HEC-1A cells and RL95-2 were co-cultured with CAFs or Rab27a-silenced CAFs for 48 h, and NEAT1 level was detected by RT-qPCR. (J)&(K) Protein levels of STAT3, p-STAT3, and YKL-40 were assessed by Western blot. (L) RT-qPCR analysis of NEAT1 level in exosomes isolated from CAFs infected with lentivirus expressing sh-NEAT1 or NEAT1 gene. (M) HEC-1A and RL95-2 cells were incubated with exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs for 48 h. RT-qPCR was performed to assess NEAT1 level. (N)&(O) STAT3 and YKL-40 expression was detected by RT-qPCR and Western blot assays. (P) The proliferation of HEC-1A and RL95-2 cells was detected by MTT assay. (Q) The growth of HEC-1A and RL95-2 cells was evaluated by colony formation assay. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot, Infection, Isolation, Incubation, Derivative Assay, MTT Assay, Colony Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Exosomal NEAT1 sponges miR-26a/b-5p to facilitate STAT3 and YKL-40 expression in EC cells. (A) RT-qPCR for miR-26a/b-5p level in exosomes derived from CAFs, NEAT1-overexpressed CAFs, or NEAT1-silenced CAFs. (B) HEC-1A and RL95-2 cells were co-cultured with NEAT1-overexpressed or silenced CAFs, and miR-26a/b-5p level was detected by RT-qPCR. (C) Expression of miR-26a/b-5p in HEC-1A and RL95-2 cells infected with lentivirus containing miR-26a/b-5p mimics or inhibitor was detected by RT-qPCR. (D) Expression of YKL-40 in HEC-1A and RL95-2 cells after overexpression or silencing of miR-26a/b-5p was assessed by Western blot. (E) The binding sites between NEAT1 and miR-26a/b-5p. Luciferase analysis (F) and RIP assay (G) was performed to determine interaction between NEAT1 and miR-26a/b-5p. The expression of STAT3 and YKL-40 in HEC-1A and RL95-2 cells received various treatments was assessed by RT-qPCR (H) and Western blot (I). All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Cell Culture, Infection, Over Expression, Western Blot, Binding Assay, Luciferase

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: MiR-26a/b-5p suppresses YKL-40 expression via targeting STAT3 in EC cells. (A) Two binding sites of miR-26a/b-5p to the 3’-UTR of STAT3. The interaction between STAT3 and miR-26a/b-5p was detected by luciferase analysis (B) and RIP assay (C). (D) The protein level of STAT3 in HEC-1A and RL95-2 cells after the transfection of STAT3 expression plasmid was assessed by Western blot. RT-qPCR (E) and Western blot (F) were used for determining YKL-40 and STAT3 expression in HEC-1A cells from various groups. (G) Three p-STAT3-binding sites (BS1, BS2, and BS3) at YKL-40 promoter were shown. (H) ChIP assay using anti-p-STAT3 antibody was used to verify the binding between p-STAT3 and the promoter of YKL-40 under exosome treatment. (I) The interaction between p-STAT3 and YKL-40 was assessed by the dual luciferase reporter assay. All data from three independent experiments were shown as mean ± SD (n = 6). **, P < 0.01; ***, P < 0.001, ns = no significant.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Expressing, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Reporter Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: CAFs-derived exosomal NEAT1 accelerates tumor growth in vivo via up-regulating YKL-40. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs. GW4869 (2 mg/kg) was intraperitoneally injected into the mice every other day. (A) The tumor growth curve was shown. (B) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (C) Immunohistochemical staining for Ki-67 expression in tumor tissues. (D) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. Nude mice were subcutaneously injected with HEC-1A cells with or without CAFs that were stably transfected with sh-NC or sh-Rab27a. (E) The tumor growth curve was shown. (F) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (G) Immunohistochemical staining for Ki-67 expression in tumor tissues. (H) Western blot analysis of YKL-40 and STAT3 protein level in tumor tissues. HEC-1A cells with or without CAFs stably transfected with sh-NEAT1 or NEAT1 gene were subcutaneously injected into the nude mice. (I) The tumor growth curve was shown. (J) RT-qPCR analysis of NEAT1 and miR-26a/b-5p expression in tumor tissues. (K) Immunohistochemical staining for Ki-67 expression in tumor tissues. (L) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Derivative Assay, In Vivo, Injection, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Western Blot, Stable Transfection, Transfection

Journal: Neoplasia (New York, N.Y.)

Article Title: Exosomal lncRNA NEAT1 from cancer-associated fibroblasts facilitates endometrial cancer progression via miR-26a/b-5p-mediated STAT3/YKL-40 signaling pathway

doi: 10.1016/j.neo.2021.05.004

Figure Lengend Snippet: Overexpression of miR-26a/b-5p reverses exosomal NEAT1-mediated protumorigenic effect in vivo . HEC-1A cells stably transfected with miR-26a/b-5p mimics or NC mimics were mixed with or without CAFs stably expressing NC vector or NEAT1, which were subcutaneously injected into the nude mice. (A) The tumor growth curve was shown. (B) Immunohistochemical staining for Ki-67 expression in tumor tissues. (C) Western blot analysis of YKL-40 and STAT3 protein levels in tumor tissues. All data from three independent experiments were shown as mean ± SD (n = 6). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To overexpress STAT3 in EC cells, the STAT3 expression plasmid (Cat: HG10034-NM, Sino Biological, Beijing, China) was transfected into EC cells using Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA).

Techniques: Over Expression, In Vivo, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Injection, Immunohistochemical staining, Staining, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Lycorine Displays Potent Antitumor Efficacy in Colon Carcinoma by Targeting STAT3

doi: 10.3389/fphar.2018.00881

Figure Lengend Snippet: Lycorine targets STAT3 in CRC Cells. (A,B) cells were treated with 40 μM of lycorine for 24 h and subsequently heated at different temperature for 3 min. after freeze-thaw cycles for cell lysis, the soluble STAT3 protein levels bound to a drug were visualized by Western blot assays. (C) Pull-down assay showing an interaction between lycorine and STAT3. Lycorine was conjugated with epoxy-activated Sepharose 6B. (D–F) Docking model of lycorine with STAT3. (D) The interaction pattern of lycorine with the residues. (E) Lycorine binding with the pocket is composed of hydrogen bonds. (F) 2D diagram between the receptor and ligand. All the western data shown are representative of at least three independent experiments.

Article Snippet: Expression vector of human STAT3 was designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Lysis, Western Blot, Pull Down Assay, Binding Assay

Journal: Frontiers in Pharmacology

Article Title: Lycorine Displays Potent Antitumor Efficacy in Colon Carcinoma by Targeting STAT3

doi: 10.3389/fphar.2018.00881

Figure Lengend Snippet: Lycorine promotes caspase-dependent mitochondrial apoptosis via STAT3 inactivation. (A , B) RKO and SW480 cells were treated with indicated concentrations of lycorine for 48 h. The protein levels of p-STAT3 were determined by Western blot assays. Total STAT3 expressions were detected as the internal control. (C–E) Cells transfected with STAT3 (STAT3 Vec) or empty vector (Control Vec) followed by lycorine treatment. (C) The protein levels of p-STAT3, STAT3, PARP, cleaved caspase-3, Bax, and Bcl-2 were detected by Western blot assays. (D) The colony formation capability was detected by colonogenic assay. (E) The cell viability was measured by MTT assay. For (E,F) , data are shown as mean ± SD ( n = 3); ∗∗ P < 0.01 compared with Vector control; ## P < 0.01 compared with Vector control transfected cells treated with lycorine (Student’s t -test). All the western data shown are representative of at least three independent experiments.

Article Snippet: Expression vector of human STAT3 was designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Western Blot, Transfection, Plasmid Preparation, MTT Assay

Journal: Frontiers in Pharmacology

Article Title: Lycorine Displays Potent Antitumor Efficacy in Colon Carcinoma by Targeting STAT3

doi: 10.3389/fphar.2018.00881

Figure Lengend Snippet: Lycorine blocks growth and development in colorectal SW480 xenograft tumors. (A–H) BALB/c nude mice were inoculated with SW480 cells and treated with lycorine or vehicle. (A) Tumors were isolated and photographed. (B) Tumors were weighted. (C) Bodies were weighted. (D) Tumor volumes were measured every 3 days. (E) PARP, cleaved caspase-3 and Bcl-2 levels were determined in xenograft tumors by Western blot assays. (F) PARP and cleaved caspase-3 expressions were determined by IHC staining in xenograft tumors. (G) p-STAT3 and total STAT3 levels were determined in xenograft tumors by Western blot assays. (H) p-STAT3 expressions were determined by IHC staining in xenograft tumors. For (B , D) , data are shown as mean ± SD ( n = 9). ∗ P < 0.05; ∗∗ P < 0.01 compared with control (Student’s t -test). For (F , H) , representative images were conducted as indicated. ∗∗∗ P < 0.001; Scale bars: 50 μm. All the western data shown are representative of at least three independent experiments.

Article Snippet: Expression vector of human STAT3 was designed and purchased from Servicebio Technologies (Wuhan, China).

Techniques: Isolation, Western Blot, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: MiR-106a-5p induces ferroptosis by targeting STAT3 in breast cancer cells. ( A ) The interaction of miR-106a-5p and STAT3 3’ UTR was identified by bioinformatic analysis using Targetscan ( http://www.targetscan.org/vert_72/ ). ( B – D ) The MDA-MB-231 and T47D cells were treated with the miR-106a-5p mimic or control mimic. ( B ) The luciferase activities of wild type STAT3 (STAT3 WT) and STAT3 with the miR-106a-5p-binding site mutant (STAT3 MUT) were determined by luciferase reporter gene assays in the cell. ( C ) The mRNA expression of STAT3 was analyzed by qPCR in the cells. ( D ) The protein expression of STAT3 and β-actin was tested by Western blot analysis in the cells. ( E ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor. The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the cells. ( E , F ) The MDA-MB-231 and T47D cells were treated with 5 mmol/L erastin, co-treated with 5 mmol/L erastin and miR-106a-5p mimic, or o-treated with 5 mmol/L erastin, miR-106a-5p mimic, and pcDNA.1-STAT3. The cell growth was analyzed by MTT assays. ( G – I ) The MDA-MB-231 and T47D cells were treated control shRNA, miR-106a-5p mimic, or co-treated with miR-106a-5p mimic and pcDNA.1-STAT3. ( G ) The levels of iron were analyzed by Iron Assay Kit. ( H ) The levels of ROS were measure by flow cytometry analysis in the cells. ( I ) The expression of GPX4, SLC7A11, and β-actin was measured by Western blot analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: Luciferase, Binding Assay, Mutagenesis, Expressing, Western Blot, shRNA, Iron Assay, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 contributes to breast cancer progression by miR-106a-5p/STAT3 axis. ( A – D ) The MDA-MB-231 and T47D cells were treated control shRNA, circRHOT1 shRNA, or co-treated with circRHOT1 shRNA and miR-106a-5p inhibitor or pcDNA.1-STAT3. ( A , B ) The cell viability was measured by MTT assays in the cells. ( C , D ) The cell apoptosis was measure by flow cytometry analysis in the cells. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: shRNA, Flow Cytometry

Journal: Aging (Albany NY)

Article Title: Circular RNA RHOT1 promotes progression and inhibits ferroptosis via mir-106a-5p/STAT3 axis in breast cancer

doi: 10.18632/aging.202608

Figure Lengend Snippet: CircRHOT1 promotes the tumor growth of breast cancer in vivo . ( A – E ) The effect of circRHOT1 on tumor growth of breast cancer cells in vivo was analyzed by nude mice tumorigenicity assay by injected with the MDA-MB-231 cells treated with control shRNA or circRHOT1 shRNA. ( A ) Representative images of dissected tumors from nude mice were presented. ( B ) The average tumor volume was calculated and shown. ( C ) The average tumor weight was calculated and shown. ( D ) The expression levels of miR-106a-5p were measured by qPCR in the tumor tissues of the mice. ( E ) The protein expression of STAT3 and β-actin was assessed by Western blot analysis in the tumor tissues of the mice. N = 5. Data are presented as mean ± SD. Statistic significant differences were indicated: ** P < 0.01.

Article Snippet: The lentiviral plasmids carrying circRHOT1 shRNA, the corresponding control shRNA, the pcDNA3.1-circRHOT1 overexpression vector, the pcDNA3.1-STAT3 overexpression vector, miR-106a-5p mimic, miR-106a-5p inhibitor, and corresponding control were synthesized and purchased (GenePharma, China) (GenScript, China).

Techniques: In Vivo, Tumorigenicity Assay, Injection, shRNA, Expressing, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Identification of osthole binding proteins. a Chemical structure of osthole intermediate and Biotin-labeled osthole. b Viability of MDA-MB-231 cells exposed to biotin-labeled osthole as determined by MTT assay. c A schematic illustrating the steps for identifying Bio-osthole binding to proteins fabricated on a microarray. d Representative image of an experimental microarray [Blue = negative control, red = positive control, yellow = positive spot]. e Magnified image of Bio-osthole binding to recombinant STAT3 protein spot on the microarray Signal to noise ratio (SNR) value is shown

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Binding Assay, Labeling, MTT Assay, Microarray, Negative Control, Positive Control, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Osthole inhibits STAT3 phosphorylation in TNBC cells. a MDA-MB-231 and BT-549 cells were treated with 200 μM osthole for the indicated times, and levels of P-STAT3 were determined by Western blot analysis. GAPDH and STAT3 were used as internal control. b MDA-MB-231 and BT-549 cells were exposed to osthole at indicated concentrations for 24 h or 12 h, respectively. P-STAT3 levels were determined by immunoblotting. GAPDH and STAT3 were used as internal control. c Cells were pretreated with 200 μM osthole for 24 h (MDA-MB-231) or 12 h (BT-549) and then stimulated with IL-6 (50 ng/mL) for 30 mins. STAT3 phosphorylation was determined by western blot. d Immunofluorescence staining of cells showing distribution of P-STAT3 (red) in MDA-MB-231 cells. DAPI was used as counter stain. e MDA-MB-231 cells were pretreated with osthole for 24 h before exposure to IL-6 (50 ng/mL) for 30 min. Nuclear extracts were subjected to P-STAT3 and STAT3 immunoblotting. Lamin B was used as loading control

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: STAT3 overexpression rescued osthole-mediated cytotoxic effects in MDA-MB-231 cells. a Representative western blot showing levels of STAT3 and P-STAT3 in MDA-MB-231 cells following transfection with STAT3 plasmid [Control plasmid = control vehicle vector, STAT3 = STAT3 plasmid]. b Quantification of STAT3 protein levels from panel A. [* P < 0.05 compared to control plasmid (V)]. c STAT3 overexpressing cells and vector control transfected cells were exposed to 200 μM osthole for 48 h, and apoptotic cells were determined by Annexin V/PI staining. d Quantification of annexin V/PI staining showing the percentage of apoptotic cells from panel C [*** P < 0.001]. (E) STAT3 overexpressing cells and vector control transfected cells were exposed to 200 μM osthole for 36 h, and representative histograms about G2/M cell cycle phase in cells were determined by flow cytometric analysis [* P < 0.05]

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: Over Expression, Western Blot, Transfection, Plasmid Preparation, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Osthole inhibits triple negative breast cancer cells by suppressing STAT3

doi: 10.1186/s13046-018-0992-z

Figure Lengend Snippet: Osthole inhibits MDA-MB-231 xenograft growth in vivo. a Tumor volume in vehicle- and osthole-treated mice. MDA-MB-231 cells were injected in the flanks of mice and tumors were allowed to develop for approximately 8 d (50–150 mm 3 ). Mice bearing MDA-MB-231 xenografts received osthole at 100 or 200 mg/kg interperitoneally [* P < 0.05]. b Images of resected tumor tissues at day 48. Two mice (one in 100 mg/kg osthole group and the other in 200 mg/kg osthole group did not have a visible tumor after 48 d treatment. c Tumor weights determined on day 48 [* P < 0.05 compared to vehicle control]. d Western blot analysis of P-STAT3 levels in resected tumor specimens. GAPDH was used as loading control. e Immunohistochemical staining of tumor sections for cell proliferation marker Ki-67, apoptosis markers Bcl-2 and cleaved caspase-3, and cell cycle markers MDM2, and CDC2. Representative images are shown

Article Snippet: Cells were transfected with expression-ready pCMV3 vector encoding STAT3 (HG10034-CF, Sino Biological, Beijing, China).

Techniques: In Vivo, Injection, Western Blot, Immunohistochemical staining, Staining, Marker

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Selective stabilization of Stat3 mRNA by Arid5a in CD4 + T cells under Th17-polarizing conditions. (A) Quantitative real-time PCR analysis of mRNAs of Stat1 , Stat3 , Stat5a , and Rorc in CD4 + T cells differentiated for 12 h under Th17 cell-inducing conditions, followed by treatment for 0–180 min with actinomycin D (ActD). (B) Luciferase activity of HEK 293T cells transfected for 48 h with the luciferase reporter vector (100 ng) encoding the STAT3 human promoter in combination with Arid5a expression vector (100–300 ng) or empty vector (100–300 ng). (C) Luciferase activity of HEK 293T cells transfected for 48 h with pGL3 vector encoding the total STAT3 3′UTR, IL-2 3′UTR, IL-17 3′UTR, or RORc 3′UTR in combination with Arid5a expression vector, empty vector, or Arid5a mutant (lacking Arid domain) vector. (D) Immunoblot analysis of Arid5a expression in HEK293T cells transfected for 48 h with Flag-Arid5a expression vector or empty vector (EV). (E) Quantitative real-time PCR analysis of mRNAs of Stat3 and Arid5a in either WT or Arid5a-deficient CD4 + T cells differentiated for 0, 1, 2, 6, 12, 24, 48, or 72 h under Th17 cell conditions. Time-course expression (0–6 h) of Stat3 and Arid5a in WT or Arid5a-deficient CD4 + T cells was enlarged (right). Data are representative of two independent experiments with similar results. (F) Quantitative real-time PCR analysis of mRNAs of Stat3 , Batf , Rorc , or Rora in either WT or Arid5a-deficient CD4 + T cells differentiated for 48 h under Th17 cell conditions. Data are representative of three independent experiments (A–D and F). Error bars show mean ± SD (A–C and F). *, P < 0.05; **, P < 0.02; ***, P < 0.001 (Student’s t test).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: The STAT3 3′UTR (1449–1792) is the responsive sites of Arid5a and an endoribonuclease Regnase-1. (A) Diagram of the region of pGL3 vector encoding STAT3 3′UTR (1–1895), STAT3 3′UTR (1–901), or STAT3 3′UTR (902–1895). The black bar shows AU-rich elements (ARE). (B) Luciferase activity of the vector encoding the STAT3 3′UTR (1–1895) in HEK293T cells transfected for 48 h together with Arid5a expression vector, Regnase-1 expression vector, or empty vector. (C) Quantitative real-time PCR analysis of mRNAs of Regnase-1 ( Reg-1 ) and Stat3 in Jurkat cells electroporated with Reg-1 siRNA or control siRNA, normalized to human Gapdh mRNA expression. (D) Quantitative real-time PCR analysis of Regnase-1 ( Reg-1 ) mRNA in Regnase-1–deficient MEFs, normalized to Gapdh mRNA expression. (E) Luciferase activity of the pGL3-vector encoding the STAT3 3′UTR (1–901) or STAT3 3′UTR (902–1895), or the pGL3-empty vector cotransfected for 48 h with Arid5a expression vector. (F) Diagram of the region of pGL3 vector encoding the STAT3 3′UTR (902–1458), STAT3 3′UTR (1449–1792), STAT3 3′UTR (1698–1895), or STAT3 3′UTR (1773–1895). The black bar shows AU-rich elements (ARE). (G) Luciferase activity of the vector encoding each area of the STAT3 3′UTR as in F cotransfected with either Arid5a expression vector or Regnase-1 expression vector. (H) Luciferase activity of the vector encoding the total STAT3 3′UTR (1–1895) in HEK293T cells for 48 h cotransfected with either Regnase-1 vector or Arid5a expression vector. Data are representative of three independent experiments (B–E, G, and H). Error bars show mean ± SD (B–E, G, and H). *, P < 0.05; **, P < 0.02; ***, P < 0.001 (Student’s t test).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Arid5a physically associates with the stem structure of the STAT3 3′UTR (1738–1765) via its residue R128. (A) A predicted conserved stem-loop region in the STAT3 3′UTR between human and mouse. Blue and red colored regions indicate stem and loop regions, respectively. (B and C) Secondary structure models of the stem-loop regions of mouse (B) and human (C) as in A. The conserved elements between human and mice are highlighted in blue. (D) Identification of the secondary structure of the STAT3 3′UTR (1738–1765) by NMR analysis. The trace of the NOESY connectivity is indicated by bold solid lines. Closed circles indicate base pairs that were experimentally identified. (E) Diagram of the stem-loop structure (1738–1765) of the STAT3 3′UTR and recombinant mouse Arid5a proteins. (F) Gel mobility shift assay (EMSA) of interaction of Arid5a recombinant protein with 3′-biotinated nucleotides as in E. (G) Docking model of Arid5a-STAT3 3′UTR (1738–1765) in silico. (H) EMSA of noninteraction of mutant Arid5a recombinant protein or interaction of Arid5a recombinant protein with 3′-biotinated nucleotides as in E. (I) Immunoblot analysis of recombinant Arid5a protein or mutant recombinant Arid5a by anti-Arid5a antibody, respectively. (J) Diagram of the stem-loop structure of the STAT3 3′UTR (1738–1765) and the mutant stem-loop structure. (K) EMSA of noninteraction of the 3′-biotinated mutant stem-loop or interaction of the 3′-biotinated stem-loop with Arid5a recombinant protein as in J. Data are representative of three independent experiments (D, F, H, I, and K).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Recombinant, Mobility Shift, In Silico, Mutagenesis, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Arid5a inhibits the binding of Regnase-1 to the stem-loop STAT3 3′UTR (1738–1765). (A) Schematic diagram of an RNA-protein binding assay. The lysates were prepared from HEK293T cells transfected with Flag-Regnase-1 expression vector or empty vector for 48 h. The 3′-biotinated nucleotides (5′-UGCAGUGGCUUGUGUUCUGGCCACUGCA-3′) of the STAT3 3′UTR (1738–1765) was conjugated with Streptavidin beads. Next, the lysates and the beads were mixed, and then washed three times. Finally, the binding proteins with the beads were eluted. (B) Immunoblot analysis of Flag-Regnase-1 protein in HEK293T cells for 48 h transfected with Flag-Regnase-1 expression vector or Flag-Regnase-1 protein binding to the stem-loop STAT3 3′UTR (1738–1765). (C) Schematic diagram of a competitive assay of RNA-protein binding. The lysates with or without Arid5a recombinant protein were prepared from HEK293T cells transfected with Flag-Regnase-1 expression vector for 48 h. Next, the lysates were mixed with the RNA-binding beads as in A, and washed three times. Finally, the binding proteins were eluted. (D) Immunoblot analysis of recombinant Arid5a protein and Flag-Regnase-1 protein in the lysates or the eluted samples, as in C. Data are representative of three independent experiments (B and D).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Binding Assay, Protein Binding, Transfection, Expressing, Plasmid Preparation, Western Blot, Recombinant, RNA Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Reduction of STAT3 level in T cells lacking Arid5a impairs a balance among STAT1, STAT3, and STAT5 activation under Th17-polarizing conditions. (A) Immunoblot analysis of STAT3, phosphorylated STAT3, and β-actin in WT or Arid5a-deficient CD4 + T cells differentiated for the indicated times toward Th17 cells. (B) Intensity of STAT3 or phosphorylated STAT3 level as in A. (C) Immunoblot analysis of STAT1, phosphorylated STAT1, and β-actin in WT or Arid5a-deficient CD4 + T cells differentiated for the indicated times toward Th17 cells. (D) Intensity of STAT1 or phosphorylated STAT1 level as in C, respectively. (E and F) Phosphorylation of STAT3 or STAT1 in WT or Arid5a-deficient CD4 + T cells for 30 min differentiated toward Th17 cells by FACS analysis. (G and H) Immunoblot analysis of Arid5a or Regnase-1 protein in CD4 + T cells differentiated for the indicated times into Th17 cells. (I) Immunoblot analysis of STAT5, phosphorylated STAT5, and β-actin in WT or Arid5a-deficient CD4 + T cells differentiated for the indicated times toward Th17 cells. (J) Intensity of STAT5 or phosphorylated STAT5 level as in I. Data are representative of three independent experiments (A–J). Data are representative from three independent experiments with similar results (B, D, and J).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

Techniques: Activation Assay, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Arid5a regulates naive CD4 + T cell fate through selective stabilization of Stat3 mRNA

doi: 10.1084/jem.20151289

Figure Lengend Snippet: Decreased STAT3 level in Arid5a-deficient T cells critically contributes to the attenuation of Th17 cell populations. (A) Immunoblot analysis of STAT3 and β-actin in WT or Arid5a-deficient CD4 + T cells for 48 h stimulated by anti-CD3ε and anti-CD28 antibodies with or without IL-6 plus TGF-β or IL-6 alone. (B and C) WT and Arid5a-deficient CD4 + T cells were transfected with MIG or MIG-STAT3C retrovirus vector for 24 h, respectively, and then cultured for 4 d under Th17-polarizing conditions. STAT3 or β-actin level in these cells was analyzed by immunoblot analysis, respectively (B). The frequency of Th17 cells in WT or Arid5a-deficient T cells by FACS analysis (C). Error bars show mean ± SD (C). (D) FACS analysis of WT or Arid5a-deficient T cells differentiated for 5 d under Th17-polarizing conditions with anti-IL-2 (JES6-1A12) treatment or isotype control (IC). (E) FACS analysis of CD4 + WT or Arid5a-deficient T cells differentiated for 5 d into Th17 cells after the stimulation of anti-CD3ε and anti-CD28 antibodies with IL-6 and IL-23 plus TGF-β (left) or IL-1β (right). Data are representative of three independent experiments (A–E).

Article Snippet: HEK293T cells were transfected with the luciferase vector encoding human STAT3 promoter (Switchgear Genomics), together with either empty vector alone or human Arid5a expression vector.

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