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: 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: European journal of cancer (Oxford, England : 1990)

Article Title: Stat3 activation is required for the growth of U87 cell-derived tumours in mice

doi: 10.1016/j.ejca.2008.11.027

Figure Lengend Snippet: Generation of stable U87 clones with hypoxia-inducible expression of DN-Stat3. (A) Bidirectional hypoxia/ HIF-responsive expression vector contains two minimal CVM promoters (CMV-MP1 and CMV-MP2) flanked by six copies of the hypoxia-responsive element (HRE) (underlined) of the human VEGF promoter placed either in left (pBI-V6L) or in right (pBI-V6R) orientations. DN-Stat3 cloned in the Not I site is driven by CMV-MP1. (B) One representative positive clone, N714, and a negative one, N715, were grown in DMEM (4.5 g glucose/l) plus 10% serum and in low glucose medium (1.0 g glucose/l) plus 5% serum, and exposed to hypoxia (1.4% O2) for 24 h. Fifty micrograms of proteins in each lane were subjected to Western blot analysis using anti-V5 antibody. (C) N705, N709, N714, N716, vector control (puromycin selected pool of 20 colonies) and parental U87 cells were exposed to 200 μM CoCl2 (mimics hypoxia) for 24 h and derived whole cell extractswere subjected to electrophoretic mobility shift assay (EMSA) using hSIE probe.

Article Snippet: Construction of DN-Stat3 expression vector Human Stat3 cDNA obtained from Robert Arceci (Johns Hopkins, Baltimore, MD, USA) was used as a template for site-directed mutagenesis to generate the DN-Stat3 construct, using the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.

Techniques: Clone Assay, Expressing, Plasmid Preparation, Western Blot, Control, Derivative Assay, Electrophoretic Mobility Shift Assay

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Stat3 activation is required for the growth of U87 cell-derived tumours in mice

doi: 10.1016/j.ejca.2008.11.027

Figure Lengend Snippet: Comparison of growth of tumours derived from DN-Stat3- expressing clones (N714 and N716) and vector control U87 cell line. U87 cells (3 × 106) (vector control or DN-Stat3 clones) in a final volume of 100 μl PBS were mixed with matrigel (1:5) and injected (subcutaneous) into right flanks of 4-week-old male nude mice. Tumour volumes were determined weekly using the formula: volume = width2 × length × 0.4 (mm3). (A) Pictures of tumour-bearing mice at week 5. (B) Kinetics of tumour growth. Each value represents mean ± SEM of 6 individual mice in each group.

Article Snippet: Construction of DN-Stat3 expression vector Human Stat3 cDNA obtained from Robert Arceci (Johns Hopkins, Baltimore, MD, USA) was used as a template for site-directed mutagenesis to generate the DN-Stat3 construct, using the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.

Techniques: Comparison, Derivative Assay, Expressing, Clone Assay, Plasmid Preparation, Control, Injection

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Stat3 activation is required for the growth of U87 cell-derived tumours in mice

doi: 10.1016/j.ejca.2008.11.027

Figure Lengend Snippet: Immunostaining for HIF-1α and DN-Stat3 in cultured cells and in DN-Stat3-derived tumours. (A) N714 cells were grown on coverslips and exposed to 200 μM CoCl2 for 48 h. Expression of DN-Stat3-V5 was observed under these conditions as green fluorescent staining by anti-V5-FITC, whereas nuclear staining of HIF-1α was detected by anti-HIF-1α (red fluorescence). Merged images are shown on the right. DAPI (blue) was used to stain nuclei. NT indicates no treatment. (B) Frozen tumour sections were fixed with ice-cold acetone. Fixed cells and tumour sections were permeabilised with PBS containing 0.3% Triron-X-100 and 3% serum, and incubated with FITC-conjugated anti-V5 antibody, washed and mounted with Vectashield containing DAPI. Discrete green fluorescent regions indicate V5 staining in the N714 clone-derived tumours. No non-specific staining was observed in either tissues from vector tumour or a no-antibody control (data not shown).

Article Snippet: Construction of DN-Stat3 expression vector Human Stat3 cDNA obtained from Robert Arceci (Johns Hopkins, Baltimore, MD, USA) was used as a template for site-directed mutagenesis to generate the DN-Stat3 construct, using the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.

Techniques: Immunostaining, Cell Culture, Derivative Assay, Expressing, Staining, Fluorescence, Incubation, Plasmid Preparation, Control

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Stat3 activation is required for the growth of U87 cell-derived tumours in mice

doi: 10.1016/j.ejca.2008.11.027

Figure Lengend Snippet: DN-Stat3 reduces tumour cell proliferation and induces apoptosis. (A) Immunohistochemical staining for Ki-67 was conducted in the paraffin-embedded, formalin-fixed tumour sections from vector- and N714 clone-derived tumours. Ki-67 was detected within the nuclei by brown stain. Haematoxylin was used for counterstaining. (B) Apoptotic cells were detected in the tumour sections by TUNEL staining. Formalin-fixed paraffin-embedded tumour (2-week post-implant) sections were de-paraffinised, hydrated and treated with proteinase K and H2O2. Then tissue sections were stained using a TUNEL assay kit. Apoptotic cells showed brown colour and counterstaining was performed with methyl green. Left panel includes representative fields from the control group consisting of sections from vector-derived and parental cell-derived tumours. Right panel shows representative fields from sections of DN-Stat3-expressing clone-derived tumours. (C) Quantification of apoptotic cells was presented as the percentage (mean ± SEM, n = 3) of TUNEL-positive cells in three random fields of each tumour of week 2 post-tumour implantation.

Article Snippet: Construction of DN-Stat3 expression vector Human Stat3 cDNA obtained from Robert Arceci (Johns Hopkins, Baltimore, MD, USA) was used as a template for site-directed mutagenesis to generate the DN-Stat3 construct, using the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.

Techniques: Immunohistochemical staining, Staining, Plasmid Preparation, Derivative Assay, TUNEL Assay, Formalin-fixed Paraffin-Embedded, Control, Expressing

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Stat3 activation is required for the growth of U87 cell-derived tumours in mice

doi: 10.1016/j.ejca.2008.11.027

Figure Lengend Snippet: DN-Stat3 prolongs the survival of mice with orthotopically implanted tumour. Kaplan–Meier curves represent survival of mice that were orthotopically implanted with U87-derived clones. (A) Vector, parental, N714 and N716 (n = 6 for each group), and (B) vector, parental, N709 and N714 (n = 8 for each group).

Article Snippet: Construction of DN-Stat3 expression vector Human Stat3 cDNA obtained from Robert Arceci (Johns Hopkins, Baltimore, MD, USA) was used as a template for site-directed mutagenesis to generate the DN-Stat3 construct, using the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Clone Assay, Plasmid Preparation

Journal: European journal of cancer (Oxford, England : 1990)

Article Title: Stat3 activation is required for the growth of U87 cell-derived tumours in mice

doi: 10.1016/j.ejca.2008.11.027

Figure Lengend Snippet: DN-Stat3 compromises neo-angiogenesis in GBM. (A) DN-Stat3 inhibits the VEGF promoter-driven luciferase activity in hypoxic U87 cells in vitro. Cells were co-transfected with a luciferase construct (2.274 kb human VEGF promoter cloned in the promoter-less luciferase expression vector, pGL3-basic) and DN-Stat3 expression plasmid. Twenty-four hour post-transfection cells were exposed to 1.4% O2 (hypoxia) or normoxia for 48 h. Derived cell extracts were subjected to luciferase assays and results were normalised. Each value of luciferase activity represents mean ± SEM of three independent determinations. (B) DN-Stat3 reduces the steady state level of VEGF mRNA in hypoxic U87 cells. Values are normalised against the β-actin level and the ratio to basal VEGF level under normoxia is shown in each case. (C) New blood vessel formation is compromised in DN-Stat3-expressing tumours. Frozen, acetone-fixed tumour sections were stained with anti-CD105 antibody followed by staining with Alexa Fluor 595, washed and mounted with Vectashield containing DAPI. Newly formed blood vessels were stained with red fluorescence, whereas nuclei were stained blue.

Article Snippet: Construction of DN-Stat3 expression vector Human Stat3 cDNA obtained from Robert Arceci (Johns Hopkins, Baltimore, MD, USA) was used as a template for site-directed mutagenesis to generate the DN-Stat3 construct, using the Quick Change XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) following the manufacturer’s instructions.

Techniques: Luciferase, Activity Assay, In Vitro, Transfection, Construct, Clone Assay, Expressing, Plasmid Preparation, Derivative Assay, Staining, Fluorescence

Journal: Biomedicines

Article Title: Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

doi: 10.3390/biomedicines10071608

Figure Lengend Snippet: Figure 2. Analysis of STAT3 in HL cell lines. (A) RQ-PCR analysis of STAT3 in eight HL cell lines (left). Western blot analysis of three HL cell lines for STAT3, phospho (P)-STAT3 and alpha-tubulin (TUBA) as control (right). (B) Immuno-cytological microscopy of untreated AM-HLH cells (left) stained for STAT3 (green) and the nucleus (blue). Forced expression of GFP-labeled STAT3 in transfected AM- HLH (right). White arrowheads indicate transfected nuclei demonstrating reduced STAT3 protein in the nucleus as compared to the cytoplasm. The light blue arrowhead indicates a non-transfected cell. (C) Western blot analysis of IL6-treated AM-HLH for STAT3, P-STAT3 and TUBA (left). Immuno- cytological microscopy of IL6-treated AM-HLH cells stained for STAT3 (green) and the nucleus (blue) (middle). RQ-PCR analysis of HLX in IL6-treated AM-HLH after 4 h (right). (D) Immuno-cytological microscopy of control and TSA-treated L-540 cells stained for STAT3 (green) and the nucleus (blue). (E) RQ-PCR analysis of HLX in L-540 (left) AM-HLH (right) treated with TSA. Statistical significance was assessed by t-test and derived p-values indicated by asterisks (** p < 0.01, *** p < 0.001, n.s., not significant).

Article Snippet: A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany).

Techniques: Western Blot, Control, Microscopy, Staining, Expressing, Labeling, Transfection, Derivative Assay

Journal: Biomedicines

Article Title: Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

doi: 10.3390/biomedicines10071608

Figure Lengend Snippet: Figure 4. Knockdown and stimulation studies in AM-HLH and L-540. (A) SiRNA-mediated knock- down of IRF4 in AM-HLH resulted in elevated expression of STAT3 as analyzed by RQ-PCR (left). The

Article Snippet: A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany).

Techniques: Knockdown, Expressing

Journal: Biomedicines

Article Title: Downregulation of STAT3 in Epstein-Barr Virus-Positive Hodgkin Lymphoma.

doi: 10.3390/biomedicines10071608

Figure Lengend Snippet: Figure 6. Summary of the results from this study showing a gene regulatory network around STAT3. Genomic amplifications targeting particular genes are highlighted by a red background. Elevated genes are indicated in red, low level expressed genes in green while medium level expressed genes are shown in black. Functional consequences including immune escape, EBV activity, proliferation and survival are highlighted by a blue background. Bold lines indicate relationships demonstrated in this study while slight lines refer to published data.

Article Snippet: A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene (Wiesbaden, Germany).

Techniques: Functional Assay, Activity Assay

Journal: Veterinary Research

Article Title: Highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses

doi: 10.1186/s13567-014-0118-3

Figure Lengend Snippet: Primer and probe sequences for quantitative reverse transcription PCR assays

Article Snippet: Primary chicken embryo cells in 6-well culture plates (Costar) were transiently transfected with constitutively active mouse STAT-3 expression plasmid [Stat3-C Flag pRc/CM V, plasmid 8722, Addgene, USA] or empty pRc/CMV vector (Invitrogen) using TransIT-LT1 reagent (Mirus Bio, Cambridge, UK).

Techniques: Reverse Transcription, Sequencing

Journal:

Article Title: Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression

doi: 10.1152/ajpgi.90493.2008

Figure Lengend Snippet: A putative Stat3 binding site regulates apelin promoter activity. Top schematic: a putative Stat3 binding site is identified in the wild-type (WT) apelin promoter sequence of the rat (−198/−195 bp); mutated nucleotides are indicated in bold in the mutated (Mut) sequence. Only partial sequences of WT and Mut promoters are shown. A: transient transfection experiments show that Stat3 overexpression increases apelin promoter activity in apelin promoter deletion-luciferase reporter constructs (−407/−1, −302/−1, −207/−1 bp). Promoter activity of the −106/−1 bp apelin promoter reporter construct was not influenced by Stat3 overexpression. B: mutation of a putative Stat3 binding site (−198/−195 bp) in the rat apelin core promoter abolished the elevation in apelin promoter activity induced by Stat3 overexpression. C: mutation of the putative Stat3 binding site inhibited the elevation in apelin promoter activity induced by LPS treatment. *P < 0.05 vs. control levels.

Article Snippet: Collagenase A was from Roche (Indianapolis, IN), and the Stat3 expression vector was from Addgene (Cambridge, MA).

Techniques: Binding Assay, Activity Assay, Sequencing, Transfection, Over Expression, Luciferase, Construct, Mutagenesis

Journal:

Article Title: Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression

doi: 10.1152/ajpgi.90493.2008

Figure Lengend Snippet: IL-6 treatment increases phosphorylated Stat3 (phospho-Stat3) levels in rat ileal cells. A: Western blotting analysis shows phospho-Stat3 or phospho-Erk1/2 levels at various times after LPS or IL-6 treatment of macrophages (RAW264.7), colon (HCT116) cells, and primary cultured rat ileal cells. Total Stat3 and Erk1/2 protein levels are also shown. LPS treatment increased phospho-Stat3 protein levels in macrophages but not in colon or primary ileal cells. IL-6 treatment increased phospho-Stat3 protein levels in primary ileal cells. LPS treatment increased phospho-Erk1/2 protein levels in colon cells and cultured rat ileal cells and in macrophages marginally. IL-6 treatment increased phospho-Erk1/2 protein levels in primary ileal cells. Equal protein loading of lanes was confirmed by reprobing blots with antibodies against total Stat3 and Erk1/2 protein levels. B: immunofluorescent staining (red fluorescence) for Stat-3 and phospho-Stat3 in primary cultured rat ileal cells. DAPI staining (blue fluorescence) indicates localization of nuclei. Stat3 is uniformly distributed throughout the cells with control treatment (B1). IL-6 treatment increases intensity of immunostained Stat3 and phospho-Stat3 in nuclei (B4, B10). Immunostained phospho-Stat3 is not detectable in control-treated cells (B7).

Article Snippet: Collagenase A was from Roche (Indianapolis, IN), and the Stat3 expression vector was from Addgene (Cambridge, MA).

Techniques: Western Blot, Cell Culture, Staining, Fluorescence

Journal:

Article Title: Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression

doi: 10.1152/ajpgi.90493.2008

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) assay indicates that IL-6 induced phospho-Stat3 is recruited to a putative Stat3 site in the rat apelin promoter in primary cultured rat ileal cells. Top: schematic of the proximal promoter region depicting location of a putative Stat3 binding site and start sites of distal and proximal primers (arrows) used in PCR amplification. −289 and −88 bp = start sites. ChIP assays were done using chromatin isolated from vehicle (lane 1) or IL-6-treated (lane 3) primary cultured rat ileal cells. Chromatin was cross-linked, sheared, and immunoprecipitated by a phospho-Stat3 antibody. Immunoprecipitations were followed by PCR amplification of the promoter region containing the putative Stat3 binding site (−198/−195 bp). Positive input DNA (lane 2) used for PCR was 2% of the amount used for immunoprecipitation. DNA size markers are shown.

Article Snippet: Collagenase A was from Roche (Indianapolis, IN), and the Stat3 expression vector was from Addgene (Cambridge, MA).

Techniques: Chromatin Immunoprecipitation, Cell Culture, Binding Assay, Amplification, Isolation, Immunoprecipitation

Journal: Frontiers in Pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: EEAC inhibits activation and lowers mRNA level of STAT3 in human HCC cells. Cells were treated with indicated concentrations of EEAC for 24 h, and then protein levels of (A) p-STAT3 and STAT3 and (B) p-JAK2 and JAK2 were determined by Western blot analyses. The relative protein levels were analyzed by Image J software. (C) mRNA levels of STAT3 (upper panel) and JAK2 (lower panel) in HepG2 and SMMC-7721 cells were detected by qRT-PCR. Data were shown as mean ± SD of three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 vs. the corresponding control.

Article Snippet: For luciferase reporter assay, cells were co-transfected with STAT3 reporter construct 4xM67 pTATA TK-Lu plus Renilla luciferase expression vector PRL-CMV (Promega, United States) using the lipofectamine 2000 reagent (Invitrogen, United States).

Techniques: Activation Assay, Western Blot, Software, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: EEAC reduces STAT3 nuclear pool and suppresses STAT3-luciferase reporter activity in human HCC cells. (A) Protein levels of STAT3 in cytoplasmic and nuclear extracts. HepG2 cells were treated with indicated concentrations of EEAC for 24 h. Protein levels of STAT3 were determined by Western blot analyses (left) and relative expression levels were analyzed by Image J software (right). GAPDH and SP1 were served as loading controls of cytoplasmic and nuclear extractions, respectively. (B) HepG2 cells were transfected with STAT3-luciferase reporter plasmid with a PRL-CMV construct for 48 h, and then treated with EEAC at indicated concentrations for another 24 h. Transcriptional activity of STAT3 was measured by the dual-luciferase reporter assay. Data were shown as mean ± SD of three independent experiments. ∗∗ P < 0.01 vs. the control.

Article Snippet: For luciferase reporter assay, cells were co-transfected with STAT3 reporter construct 4xM67 pTATA TK-Lu plus Renilla luciferase expression vector PRL-CMV (Promega, United States) using the lipofectamine 2000 reagent (Invitrogen, United States).

Techniques: Luciferase, Activity Assay, Western Blot, Expressing, Software, Transfection, Plasmid Preparation, Construct, Reporter Assay

Journal: Frontiers in Pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: EEAC downregulates protein levels of STAT3-targeted molecules. HepG2 (A) and SMMC-7721 (B) cells were treated with indicated concentrations of EEAC for 24 h, and then protein levels of Bcl-2, Bcl-xL, MMP-2 and MMP-9 were determined by immunoblotting. The relative protein levels were analyzed by Image J software and shown as mean ± SD of three independent experiments. ∗ P < 0.05, ∗∗ P < 0.01 vs. the corresponding control.

Article Snippet: For luciferase reporter assay, cells were co-transfected with STAT3 reporter construct 4xM67 pTATA TK-Lu plus Renilla luciferase expression vector PRL-CMV (Promega, United States) using the lipofectamine 2000 reagent (Invitrogen, United States).

Techniques: Western Blot, Software

Journal: Frontiers in Pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: Over-activation of STAT3 in HepG2 cells diminishes the cytotoxic effects of EEAC. (A) Protein levels of STAT3 and p-STAT3. HepG2 cells were transiently transfected with either an empty vector or an STAT3C-expressing construct for 48 h, and then total cell lysates were extracted for Western blot analyses. (B) After transfection, HepG2 cells were treated with EEAC for 48 h, the cell viability was determined by the MTT assay. Data were shown as mean ± SD of three independent experiments. ∗ P < 0.05 vs. cells transfected with the empty vector.

Article Snippet: For luciferase reporter assay, cells were co-transfected with STAT3 reporter construct 4xM67 pTATA TK-Lu plus Renilla luciferase expression vector PRL-CMV (Promega, United States) using the lipofectamine 2000 reagent (Invitrogen, United States).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Construct, Western Blot, MTT Assay

Journal: Frontiers in Pharmacology

Article Title: Antrodia camphorata Mycelia Exert Anti-liver Cancer Effects and Inhibit STAT3 Signaling in vitro and in vivo

doi: 10.3389/fphar.2018.01449

Figure Lengend Snippet: EEAC suppresses tumor growth in SMMC-7721 cell-bearing mice. Nude mice bearing SMMC-7721 xenograft were treated with EEAC for 18 days. (A) Tumor volume. (B) Representative tumors removed from mice. Tumor weights were recorded. In (A,B) , data were presented as mean ± SEM, n = 6. (C) TUNEL assays for apoptosis in tumor tissues collected from three individual mice. (D) Western blot analyses for protein levels of p-STAT3, STAT3, p-JAK2 and JAK2 (left panel) and the relative protein levels were analyzed by Image J software (right panel). Data were shown as mean ± SEM, n = 3. ∗ P < 0.05, ∗∗ P < 0.01 vs. vehicle control.

Article Snippet: For luciferase reporter assay, cells were co-transfected with STAT3 reporter construct 4xM67 pTATA TK-Lu plus Renilla luciferase expression vector PRL-CMV (Promega, United States) using the lipofectamine 2000 reagent (Invitrogen, United States).

Techniques: TUNEL Assay, Western Blot, Software