Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Targeting stromal androgen receptor suppresses prolactin-driven benign prostatic hyperplasia (BPH).

doi: 10.1210/me.2013-1207

Figure Lengend Snippet: Figure 5. Elevated GM-CSF expression from PRL-treated BPH-1-PRLR cells enhances the stromal cell growth through sustaining STAT3 activity, and stromal AR is required to modulate such epithelial-stromal interaction. A, Stromal cells (pshTERT, left panel; and WPMY-1, middle panel) were treated with CMs from BPH-1-V or BPH-1-PRLR treated with or without 100 ng/mL hPRL for 48 hours. The standard MTT cell growth assay was conducted at days 0, 2, 4, and 6. The data are presented as mean SD from at least 3 independent experiments. Right panel, The cartoon depicts the coculture system of epithelial and stromal cells. B, The BPH-1-PRLR cells were treated with or without 100 ng/mL hPRL for 48 hours, and the CMs were harvested for human growth factor protein array analyses. The rectangles point out the increased expression of G-CSF and GM-CSF in BPH-1PRLR hPRL cells. C, The RNAs were harvested from BPH-1-V and BPH-1-PRLR treated with or without 100 ng/mL hPRL for 8 hours and subjected to Q-PCR analysis. Each bar represents the mean SD of at least 2 independent determinations performed in triplicate. D, The exogenous GM-CSF can increase pshTERT stromal cell growth. The siLuc and siAR pshTERT stromal cells were cultured with media containing 10 ng/mL or 50 ng/mL human GM-CSF recombinant proteins. The media were replaced every other day and subjected to MTT cell growth assay at days 0, 2, 4, and 6. Each bar represents the mean SD of at least 2 independent determinations performed in triplicate. E, The addition of neutralizing GM-CSF antibody in the CMs of hPRL treated BPH-1-PRLR cells inhibits the pshTERT cell growth. The siLuc and siAR pshTERT stromal cells were cultured under the CMs (BPH-1-PRLR hPRL) with IgG or 10 g/mL anti-GM- CSF antibody. F, The cell growth assay from siLuc-pshTERT and siAR-pshTERT cells cocultured with the CMs from BPH-1-PRLR hPRL was measured. The selective STAT3 inhibitor, 50 M NSC74859, was added to the CMs. ***, P .001 (siLuc pshTERT CM vs siAR pshTERT CM and siLuc pshTERT CM vs siLuc pshTERT CM 50 M NSC74859). G, The cell growth assay from siLuc/shRNA control (ctl), siLuc/STAT3 shRNA, siAR/shRNA control, and siAR/ STAT3 shRNA knockdown pshTERT cells with culture media containing 50 ng/mL GM-CSF was measured. **, P .01 (siLuc/shRNA ctl pshTERT vs siLuc/ STAT3 shRNA pshTERT). *, P .05 (siAR/shRNA ctl pshTERT vs siAR/STAT3 shRNA pshTERT). In panels D–G, each bar represents the mean SD of at least 2 independent determinations performed in triplicate. *, P .05; **, P .01; ***, P .001.

Article Snippet: The antibodies against AR (C-19, N-20; Santa Cruz Biotechnology), smooth muscle -actin (SMA; Sigma-Aldrich), signal transducer and activator of transcription (STAT)-5 (Cell Signaling), phosphorylated (p)-STAT5 at Tyr 694 (Cell Signaling), STAT3 (Cell Signaling), p-STAT3 at Tyr 705 (Cell Signaling), protein inhibitor of activated STAT3 (PIAS)-3 (Abcam), -tubulin (Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology), actin (Santa Cruz Biotechnology), CD3 (Sigma-Aldrich), B220 (BD Biosciences), and procollagen 1A (Santa Cruz Biotechnology) were used.

Techniques: Expressing, Activity Assay, Growth Assay, Protein Array, Cell Culture, Recombinant, shRNA, Control, Knockdown

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: Targeting stromal androgen receptor suppresses prolactin-driven benign prostatic hyperplasia (BPH).

doi: 10.1210/me.2013-1207

Figure Lengend Snippet: Figure 7. The proposed working models of cross talking signals between epithelial PRL/PRLR-STAT5 and stromal GM-CSF/GM-CSFR- STAT3-AR. The illustration depicts the interacting signaling between hyperplastic epithelium and stromal cells in Pb-PRL tg mice and the in vitro coculture system. The constitutive epithelial PRL autonomously activates the PRL/PRLR-STAT5 signal cascade in hyperplastic epithelium and therefore enhances the GM-CSF expression. The GM-CSF binds to GM-CSFR in the stromal cells to facilitate cells growth through sustaining STAT3 activity. In the Wt-AR/Pb-PRL() mice or siLuc pshTERT stromal cells, AR can interact with PIAS3 and release STAT3 to the nucleus to turn on STAT3 target genes and facilitate the cell growth. In contrast, in the absence of stromal AR, PIAS3 in dARKO/Pb- PRL() mice or siAR pshTERT stromal cells can suppress STAT3 activity via associating with STAT3. GM, granulocyte macrophage colony- stimulating factor (CSF). GMR, granulocyte macrophage CSF receptor-. c, granulocyte macrophage CSF receptor chain.

Article Snippet: The antibodies against AR (C-19, N-20; Santa Cruz Biotechnology), smooth muscle -actin (SMA; Sigma-Aldrich), signal transducer and activator of transcription (STAT)-5 (Cell Signaling), phosphorylated (p)-STAT5 at Tyr 694 (Cell Signaling), STAT3 (Cell Signaling), p-STAT3 at Tyr 705 (Cell Signaling), protein inhibitor of activated STAT3 (PIAS)-3 (Abcam), -tubulin (Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology), actin (Santa Cruz Biotechnology), CD3 (Sigma-Aldrich), B220 (BD Biosciences), and procollagen 1A (Santa Cruz Biotechnology) were used.

Techniques: In Vitro, Expressing, Activity Assay

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 1. Expression of signal transducers and activators of transcription 3 (STAT3) in SW1990 cells and HeLa cells. (a) STAT3 expression was determined by Western blot analysis. Detection of β-actin was used as a loading control. HeLa cell line served as a positive control of STAT3 expression. (b) STAT3 distribution in SW1990 cells was examined by immunocytochemistry (400 ×), the buffy particle corresponded for STAT3 (shown by arrow).

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Western Blot, Control, Positive Control, Immunocytochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 2. Effects of signal transducers and activators of transcription 3 (STAT3) specific shRNA expression vector on STAT3 expression. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3- siRNA-II and pRNAT-STAT3-siRNA-III were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT- PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with pRNAT-Con, pRNAT-STAT3-siRNA-I, pRNAT-STAT3-siRNA-II and pRNAT-STAT3-siRNA-III. The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was deter- mined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. Results shown are for one represent- ative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: shRNA, Expressing, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Membrane, Control

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 3. Effects of stable transfection of pRNAT-STAT3-siRNA-II vector on signal transducers and activators of transcription 3 (STAT3) expres- sion. (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for STAT3 and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole and nuclear protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of STAT3 protein was determined using Western blot analysis with an anti-STAT3 antibody. The expression of p-STAT3 protein was determined by hybridizing the same membrane filter with an antip-STAT3 antibody. The levels of β-actin expression were determined as a control for equivalent protein loading. (c) STAT3 DNA-binding activity. Cell nuclear protein extracts (10 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) and subjected to electrophoretic mobility shift analysis (EMSA) using biotin end-labeled oligonucleotide probes containing a consensus-binding motif for STAT3. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Stable Transfection, Plasmid Preparation, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Western Blot, Expressing, Membrane, Binding Assay, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 4. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell invasion in vitro. SW1990-RNAi-a, SW1990-RNAi-b, SW1990-Con and the parental cell lines were seeded into the upper compart- ments of invasion chambers. Cells were allowed to invade for 48 h at 37°C. The tumor cells that invaded the ECMatrix and migrated through the polycarbonate membrane were stained by the staining solution and dissolved in 10% acetic acid. The dye/solution mixture was transferred onto a 96-well plate for colorimetric reading of optical density (OD) at 560 nm. The number of migrated cells that penetrated through the ECMatrix-coated filters was expressed as the OD at 560 nm. The standard deviation bars represent replicates within the assay. *P < 0.001 compared with that of parental SW1990 cells. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vitro, Membrane, Staining, Standard Deviation

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 5. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on SW1990 cell metastasis in vivo. In total, 5 × 105 SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were injected i.v. into groups of nude mice (n = 6). The mice were killed on day 21 or when mice became moribund. The number of experimental lung metastases was determined with the aid of a dissecting microscope. Results were expressed as the median number and range of lung metastasis nodules. *P < 0.001 compared with that of parental SW1990 cells. Results shown here are for one representative experiment of three. (a) H&E staining of formalin-fixed, paraffin-embedded lung tissue of nude mice (100 ×). The metastasis nodus are indicated by arrows. (b) H&E staining of formalin-fixed, paraffin-embedded metastasis nodus of lung tissue of nude mice (400 ×). (c) In SW1990 cells injected nude mice, STAT3, matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF) distribution in metastasis nodus of lung tissue was examined by immunohistochemistry (400 ×), the buffy particle corresponded for STAT3, MMP-2 and VEGF. Immunohistochemistry showed that STAT3, MMP-2 and VEGF were distributed in cytoplasm.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: In Vivo, Transfection, Control, Plasmid Preparation, Injection, Microscopy, Staining, Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Cancer science

Article Title: RNA interference-mediated signal transducers and activators of transcription 3 gene silencing inhibits invasion and metastasis of human pancreatic cancer cells.

doi: 10.1111/j.1349-7006.2007.00485.x

Figure Lengend Snippet: Fig. 6. Effects of silence of signal transducers and activators of transcription 3 (STAT3) gene on the expression of matrix metalloproteinases-2 (MMP-2) and vascular endothelial growth factor (VEGF). (a) Reverse transcription-polymerase chain reaction (RT-PCR) analysis. The RNA samples (2 µg in each) extracted from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3- RNAi (SW1990-RNAi-a and SW1990-RNAi-b) were subjected to RT-PCR for MMP-2, VEGF, and β-actin mRNAs as described in Materials and Methods. RT-PCR for β-actin was carried out in parallel to show an equal amount of total RNA in the sample. (b) Western blot analysis. Cellular whole protein extracts (100 µg in each) were prepared from SW1990 cells, SW1990 cells transfected with a control vector (SW1990-Con), and SW1990 cells transfected with STAT3-RNAi (SW1990-RNAi-a and SW1990-RNAi-b). The expression of MMP-2 protein was determined using Western blot analysis with an anti-MMP-2 antibody. The expression of VEGF protein was determined by hybridizing the same membrane filter with an anti-VEGF antibody. The levels of β-actin expression were determined as a control for the equivalent protein loading. Results shown are for one representative experiment of three.

Article Snippet: Membranes were incubated in blocking buffer (1 × TBS, 0.1% Tween 20, and 5% non-fat dry milk) for 1 h at room temperature, followed by hybridization with antip-STAT3[tyr-705] antibody (Cell Signal, 1:1000 dilution), anti-STAT3 antibody (Cell Signal, 1:1000 dilution), anti-MMP-2 antibody (Santa Cruz, 1:500 dilution), anti-VEGF antibody (Santa Cruz, 1:500 dilution) or antiβ-actin antibody (Labvision, Fremont, CA, USA, 1:100 dilution), at 4°C overnight.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Western Blot, Membrane

Journal: Oncotarget

Article Title: Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer

doi:

Figure Lengend Snippet: Western blot analysis of pSTAT3 protein levels in HUVECs and PC3M-1E8 cells, representative snap-frozen primary prostate tumor tissues (T) and matched adjacent nontumor tissues (N).

Article Snippet: Standard western blot analysis was performed with antibodies for STAT3, pSTAT3 (Tyr705), vascular endothelial growth factor (VEGF), survivin, c-Myc, Nanog, Oct-4, β-actin (Santa Cruz, CA) and Bcl-xL (Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer

doi:

Figure Lengend Snippet: (A) PC3M-1E8 cells were treated with different doses of Stattic for 4 h. (B) PC3M-1E8 cells were treated with 10 μM Stattic for different time points. (C) Western blot analysis of STAT3 downstream targets in PC3M-1E8 cells treated with 10 μM Stattic for 24 h. (D) Cellular localization of pSTAT3 and survivin (green) was observed by confocal microscopy after immunofluorescent staining of PC3M-1E8 cancer cells treated with 10 μM Stattic. (E) PC3M-1E8 cells were cultured in serum-free medium overnight, then treated with 25 ng/mL IL-6 and various amounts of Stattic for 24 hours. (F) PC3M-1E8 cells in serum-free medium were treated with 25 ng/mL IL-6 and 10 μM Stattic and examined for pSTAT3 and survivin expression by immunofluorescence (green). (G) PC3M-1E8 cells were treated with different doses of Stattic. After 48 h, morphological examinations were performed. (H) PC3M-1E8 cells were treated with different doses of Stattic. After 48 h, cells were stained with acridine orange for visualizing apoptotic cells. (I) A2780 ovarian cancer cells and HUVECs were treated with 20 μM Stattic. After 48 h, morphological examinations were performed. (J) A2780 cells and HUVECs were treated with 20 μM Stattic. After 48 h, cells apoptosis was analyzed by flow cytometry. (K) PC3M-1E8 cells were treated with 10 μM Stattic in the absence or presence of 25 ng/mL IL-6. After 48 h, cells apoptosis was analyzed by flow cytometry.

Article Snippet: Standard western blot analysis was performed with antibodies for STAT3, pSTAT3 (Tyr705), vascular endothelial growth factor (VEGF), survivin, c-Myc, Nanog, Oct-4, β-actin (Santa Cruz, CA) and Bcl-xL (Cell Signaling Technology, Danvers, MA).

Techniques: Western Blot, Confocal Microscopy, Staining, Cell Culture, Expressing, Immunofluorescence, Flow Cytometry

Journal: Oncotarget

Article Title: Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer

doi:

Figure Lengend Snippet: (A) Representative images of soft agar assay (left panel). Quantitation of colonies from soft agar assay of PC3M-1E8 cells treated with Stattic (right panel). (B) PC3M-1E8 cells were subjected to ALDEFLUORH assay in order to identify cells with high ALDH expression (ALDH high ). The ALDH inhibitor DEAB was used as a negative control (left panel). The cells without inhibitor shifted to the right were considered ALDH high cells (right panel). (C) ELISA for pSTAT3 in ALDH high subpopulations and non-ALDH high subpopulations derived from PC3M-1E8 cells and primary prostate cancer cells. (D) Western blot analysis of ALDH high cells derived from PC3M-1E8 cells treated with 10 μM Stattic for 4 h. (E) Stattic significantly decreased the proportion of ALDH high prostate cancer cells in vitro . (F) Western blot analysis of PC3M-1E8 cells treated with 10 μM Stattic for 4 h. (G) Number of ALDH high cells formed from non-ALDH high cells obtained by sorting PC3M-1E8 cells and primary prostate cancer cells upon treatment with IL-6 in the presence or absence of Stattic. (H) Colony formation ability of STAT3 knock-down PC3M-1E8 cells treated with 25 ng/ml IL-6. Quantitation of colonies that were at least 20 μm in diameter was recorded. * P < 0.05, ** P <0.01.

Article Snippet: Standard western blot analysis was performed with antibodies for STAT3, pSTAT3 (Tyr705), vascular endothelial growth factor (VEGF), survivin, c-Myc, Nanog, Oct-4, β-actin (Santa Cruz, CA) and Bcl-xL (Cell Signaling Technology, Danvers, MA).

Techniques: Soft Agar Assay, Quantitation Assay, Expressing, Negative Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, In Vitro, Knockdown

Journal: Oncotarget

Article Title: Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer

doi:

Figure Lengend Snippet: (A) PC3M-1E8 cells were transduced with a GFP lentivirus and examined by fluorescence microscopy 72 hours later. (B) Western blot analysis shows that STAT3, pSTAT3 and VEGF were downregulated in PC3M-1E8 cells transduced with STAT3 shRNA. (C) VEGF analysis by ELISA. (D) Immunofluorescence staining of pSTAT3 (red) on PC3M-1E8 cells transduced with STAT3 shRNA (E) Representative diagram of the coculture assay. (F) Representative images of cocultured HUVECs. (G) HUVECs proliferation was measured through MTT assay. (H and I) The effects of conditioned medium from PC3M-1E8 cells transduced with STAT3 shRNA on angiogenesis in vitro . Representative images are displayed. * P < 0.05, ** P <0.01.

Article Snippet: Standard western blot analysis was performed with antibodies for STAT3, pSTAT3 (Tyr705), vascular endothelial growth factor (VEGF), survivin, c-Myc, Nanog, Oct-4, β-actin (Santa Cruz, CA) and Bcl-xL (Cell Signaling Technology, Danvers, MA).

Techniques: Transduction, Fluorescence, Microscopy, Western Blot, shRNA, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Co-culture Assay, MTT Assay, In Vitro

Journal: Oncotarget

Article Title: Inhibition of STAT3 signaling targets both tumor-initiating and differentiated cell populations in prostate cancer

doi:

Figure Lengend Snippet: (A) Representative images of prostate tumor from nude mice that received subcutaneous injections of PC3M-1E8 cells infected with STAT3 shRNA-expressing lentivirus or control lentivirus (left panel). Tumor volumes were measured after tumor cell inoculation every 7 days for a period of 4 weeks (right panel). Error bar represents SD (n = 5). (B) Immunoblots of pSTAT3, c-Myc and Oct4 in xenograft tumors at the end of the experiment. (C) Measurement of subcutaneous xenograft tumor size after treatment with Stattic. Error bar represents SD (n = 5). (D) Tumor bearing mice (n=3) were sacrificed after 2nd, 5th and 9th injection to assess tumor growth. (E) Immunoblots of pSTAT3, c-Myc and Bcl-xL in xenograft tumors treated with DMSO or Stattic on day 28 following first treatment. Representative data from 1 of 3 independent experiments are shown. (F) Xenograft tumors (n=3) were subjected to enzymatic dissociation, ALDEFLUOR staining and flow cytometry assay on day 28 following first treatment. (G) Immunoblots of pSTAT3 in xenograft tumors treated with DMSO or Stattic on day 28 following first treatment. Each lane represents the tumor lysate of an individual experimental mouse. Representative data from 1 of 3 independent experiments are shown. (H) TGI of Stattic in the F3 generation of three patient tumors. Five mice were used in each experimental group. (I) Patient-derived xenograft tumors (n=5) were subjected to enzymatic dissociation, ALDEFLUOR staining and flow cytometry assay on day 28 following first treatment. (J~L) Kaplan-Meier survival curves of mice (n = 8 for each group) implanted with human primary prostate tumors after Stattic treatment. The log-rank test was used to determine differences between Kaplan-Meier curves. * P < 0.05, ** P <0.01.

Article Snippet: Standard western blot analysis was performed with antibodies for STAT3, pSTAT3 (Tyr705), vascular endothelial growth factor (VEGF), survivin, c-Myc, Nanog, Oct-4, β-actin (Santa Cruz, CA) and Bcl-xL (Cell Signaling Technology, Danvers, MA).

Techniques: Infection, shRNA, Expressing, Control, Western Blot, Injection, Staining, Flow Cytometry, Derivative Assay

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: HBV activates STAT3 signaling which backs to promote HBV virus replication. (A) Western blotting analysis of STAT3 and p-STAT3 levels in HepG2.2.15 and HepG2 cells. (B) Western blotting analysis of STAT3 and p-STAT3 levels in HBV-stably transfected HepG2 and HL7702 cells. (C) Western blotting analysis of STAT3 and p-STAT3 levels in HepG2.2.15 cells incubated with 100 ng/mL IL-6. (D) After 6 h or 24 h, mRNA levels of HBx was analyzed by qRT-PCR in HepG2.2.15 cells incubated with 100 ng/mL IL-6. Data are expressed as the mean ± SEM from at least 3 separate experiments. *p < 0.05, **p < 0.01.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Western Blot, Stable Transfection, Transfection, Incubation, Quantitative RT-PCR

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: HBV virus replication was inhibited by STAT3-Decoy ODN. After transfected with STAT3-Decoy ODN for 48 h, HBx mRNA in HepG2.2.15 cells and supernatant HBV DNA were detected by qRT-PCR (A), and HBsAg and HBeAg protein levels were detected by ELISA (B). Data are expressed as the mean ± SEM from at least 3 separate experiments. *p < 0.05, **p < 0.01.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: HBV virus replication was inhibited by STAT3-shNRAs. After HepG2.2.15 cells transfected with STAT3-shRNA or LMP control plasmid for 48 h, the expression of STAT3 was detected by qRT-PCR (left) and western blotting (right) (A), HBx mRNA in HepG2.2.15 cells and supernatant HBV DNA were detected by qRT-PCR (B), and HBsAg and HBeAg protein expression were detected by ELISA (C). Data are expressed as the mean ± SEM from at least 3 separate experiments. *p < 0.05, **p < 0.01.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Transfection, shRNA, Plasmid Preparation, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: The growth of HBV positive HCC cells was inhibited by blocking STAT3 signaling. HepG2.2.15 cells were transfected with STAT3-shRNA for 48 h, (A) the proliferation of HepG2.2.15 cells was measured by MTT assay, (B) the apoptosis of HepG2.2.15 cells were analyzed by Annexin V and PtdIns staining method, and (C) the cell cycle analysis was subjected to flow cytometry. Data are expressed as the mean ± SEM from at least 3 separate experiments. *p < 0.05, **p < 0.01.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Blocking Assay, Transfection, shRNA, MTT Assay, Staining, Cell Cycle Assay, Flow Cytometry

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: HBV- induced angiogenesis was inhibited by STAT3-shRNAs. (A) VEGF, MMP-9 and TGF-β mRNA in HepG2.2.15 and HepG2 cells were detected by qRT-PCR. (B) HUVECs seeded on matrigel were treated with medium, supernatant of HepG2 and HepG2 2.15 cells for 4 h, and then the network growth area was examined using an inverted fluorescence microscope (original magnification = 200×). Tube formation was digitally quantified by assessing the number of tubes (n). (C) After transfected with STAT3-shRNA for 48h, the relative expression of VEGF, MMP-9 and TGF-β was analyzed in HepG2.2.15 cells. (D) HUVECs seeded on matrigel were treated with supernatant of HepG2.2.15 cells transfected with STAT3-shRNAs or control LMP, and then tube formation was digitally quantified by assessing the number of tubes (n). Representative images and statistical data are expressed as the mean ± SEM from at least 3 separate experiments. *p < 0.05, **p < 0.01.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Quantitative RT-PCR, Fluorescence, Microscopy, Transfection, shRNA, Expressing

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: STAT3-shRNAs exhibited anti-HBV positive HCC role in xenograft mice. Following subcutaneously (s.c.) injection of HepG2.2.15 cells, these nude mice bearing palpable tumor were intratumorally treated with STAT3-shRNAs on day 10,2 and 15, and tumor tissue (A) and statistical analysis of tumor volume (B) were shown. (C-D) The volume of tumor (C) and the weight of tumor (D) were measured at the indicated time. Results were shown as means ± s.e.m., n = 4. *p < 0.05, **p < 0.01.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Injection

Journal: Cancer Biology & Therapy

Article Title: Targeting blockage of STAT3 inhibits hepatitis B virus-related hepatocellular carcinoma

doi: 10.1080/15384047.2016.1156257

Figure Lengend Snippet: Primer sequences used in the real-time PCR assays.

Article Snippet: Human STAT3 shRNAs were designed using BLOCK-iTTM RNAi Designer (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Oncotarget

Article Title: MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway

doi:

Figure Lengend Snippet: A. , (left) a representative result of Western blotting analysis shows the expression levels of IL-8 and phosphor-Stat3 in the control or miR-23a agomir-injected CNE2-IR xenografts; (right) a histogram shows the average levels of IL-8 and phosphor-Stat3 in the tumors ( n = 5 each group). B. , a representative result of Western blotting analysis shows phospho-Stat3 levels in the IL-8-stimulated, miR-23a inhibitor-transfected, or miR-23a inhibitor and IL-8 shRNA 1-cotransfected CNE2 cells, and IL-8 knockdown, miR-23a mimic-transfected or miR-23a mimic-transfected and IL-8-stimulated CNE2-IR cells as well as their corresponding controls. C. , a representative result of immunofluorescent staining shows the nuclear translocation of phospho-Stat3 in the IL-8-stimulated, miR-23a inhibitor-transfected, or miR-23a inhibitor and IL-8 shRNA 1-cotransfected CNE2 cells, and IL-8 knockdown, miR-23a mimic-transfected or miR-23a mimic-transfected and IL-8-stimulated CNE2-IR cells as well as their corresponding controls. D. , Stat3 luciferase reporter activity in the miR-23a inhibitor-transfected or IL-8-stimulated CNE2 cells, and miR-23a mimic-transfected or IL-8 knockdown CNE2-IR cells. Means, SDs, and statistical significance are denoted; **, P < 0.01.

Article Snippet: Immunofluorescent staining was performed to detected the nuclear translocation of phospho-Stat3 in the NPC cells using anti-phospho-Stat3 (Tyr705) antibody (#9145, CST) and secondary antibody conjugated with Alexa Fluor 594 (Vector Laboratories) as previously described by us [ ].

Techniques: Western Blot, Expressing, Control, Injection, Transfection, shRNA, Knockdown, Staining, Translocation Assay, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway

doi:

Figure Lengend Snippet: A. , a clonogenic survival assay shows that inhibition of Stat3 activity decreased CNE2-IR cell radioresistance. CNE2-IR cells treated with 5 µmol/L Stattic or vehicle were irradiated with a range of 1-8Gy radiation doses, and dose survival curves were created by fitting surviving fractions to the linear quadratic equation. B. , a clonogenic survival assay shows that Stat3 overexpression (OE) increased CNE2 cell radioresistance. CNE2-IR cells transfected with 4 μg/mL Stat3 expression or control vector were irradiated with a range of 1-8Gy radiation doses, and dose survival curves were created by fitting surviving fractions to the linear quadratic equation. C. , a clonogenic survival assay shows that inhibition of Stat3 signaling significantly abolished CNE2 cell radioresistance induced by exogenous IL-8 stimulation. CNE2 cells treated with 5 µmol/L Stattic and 4.5 ng/mL IL-8 were irradiated with a range of 1-8Gy radiation doses, and dose survival curves were created by fitting surviving fractions to the linear quadratic equation. D. , a representative image of IL-8 and phospho-Stat3 immunohistochemical staining in the normal nasopharyngeal mucosal tissue (a), radiosensitive NPC tissue (b) and radioresistant NPC tissue (c). Original magnification, ×200. Means, SDs, and statistical significance are denoted.

Article Snippet: Immunofluorescent staining was performed to detected the nuclear translocation of phospho-Stat3 in the NPC cells using anti-phospho-Stat3 (Tyr705) antibody (#9145, CST) and secondary antibody conjugated with Alexa Fluor 594 (Vector Laboratories) as previously described by us [ ].

Techniques: Clonogenic Cell Survival Assay, Inhibition, Activity Assay, Irradiation, Over Expression, Transfection, Expressing, Control, Plasmid Preparation, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway

doi:

Figure Lengend Snippet: The levels of IL-8 and phospho-Stat3 in the nasopharygeal carcinoma and normal nasopharyngeal mucosal tissues

Article Snippet: Immunofluorescent staining was performed to detected the nuclear translocation of phospho-Stat3 in the NPC cells using anti-phospho-Stat3 (Tyr705) antibody (#9145, CST) and secondary antibody conjugated with Alexa Fluor 594 (Vector Laboratories) as previously described by us [ ].

Techniques:

Journal: Oncotarget

Article Title: MiR-23a sensitizes nasopharyngeal carcinoma to irradiation by targeting IL-8/Stat3 pathway

doi:

Figure Lengend Snippet: The correlations shown include those between IL-8 and phospho-Stat3 A. , IL-8 and miR-23a B. and phospho-Stat3 and miR-23a C. .

Article Snippet: Immunofluorescent staining was performed to detected the nuclear translocation of phospho-Stat3 in the NPC cells using anti-phospho-Stat3 (Tyr705) antibody (#9145, CST) and secondary antibody conjugated with Alexa Fluor 594 (Vector Laboratories) as previously described by us [ ].

Techniques:

Journal: Oncotarget

Article Title: Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

doi: 10.18632/oncotarget.7051

Figure Lengend Snippet: ( A ) Structure of physalin A. ( B ) The human NSCLC cell lines, H292, H358, H1975, H460, A549, and BEAS-2B (1 × 10 4 cells/well) were treated with the indicated concentrations of physalin A for 24 h. Cell viability was then measured using the CCK-8 assay. Results are presented as mean ± SD from three independent experiments. (CB) p-STAT3 (Tyr 705) and STAT3 levels were detected in the H292, H358, H1975, H460 and A549 cell lines. β-actin was used as a loading control.

Article Snippet: STAT3 small interfering RNA (sense: 5′-GGGACCUGGUGUGAAUUAUTT-3′, antisense: 5′-AUAAUUCACACCAGGUCCCTT-3′) and scrambled control siRNA (sense: 5′-UUCU CCGAACGUG UCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGA GAATT-3′) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: CCK-8 Assay, Control

Journal: Oncotarget

Article Title: Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

doi: 10.18632/oncotarget.7051

Figure Lengend Snippet: ( A ) Physalin A (PA) inhibits STAT3 phosphorylation at tyrosine 705 but not at serine 727 in a dose-dependent manner. Cells were treated with the indicated concentration of PA for 4 h, and p-STAT3 (Tyr 705), p-STAT3 (Ser 727) and STAT3 levels were determined by Western blot analysis. β-actin was used as an internal loading control. ( B ) Cells were pretreated with PA for 6 h followed by 25 ng/mL of IL-6 to induce STAT3 tyrosine 705 phosphorylation. Cell lysates were subjected to Western blot analysis using antibodies specific for p-STAT3 (Tyr 705), p-STAT3 (Ser727), p-STAT3, STAT3 and β-actin.

Article Snippet: STAT3 small interfering RNA (sense: 5′-GGGACCUGGUGUGAAUUAUTT-3′, antisense: 5′-AUAAUUCACACCAGGUCCCTT-3′) and scrambled control siRNA (sense: 5′-UUCU CCGAACGUG UCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGA GAATT-3′) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Concentration Assay, Western Blot, Control

Journal: Oncotarget

Article Title: Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

doi: 10.18632/oncotarget.7051

Figure Lengend Snippet: ( A ) H292 cells were treated with scrambled siRNA, STAT3 siRNA, 10 μM physalin A (PA), STAT3 siRNA+ 10 μM PA or scrambled siRNA+ 10 μM PA. Cell apoptosis was determined by flow cytometry using an Annexin V-FITC/PI staining kit. ( B ) Cell lysates were isolated for Western blot analysis to detect p-STAT3 (Tyr 705) and STAT3. β-actin was used as an internal loading control. *, †, ‡, §, || p < 0.05, significantly different from the *control, † scrambled siRNA, ‡ STAT3 siRNA, § 10 μM PA and || STAT3 siRNA + 10 μM PA groups.

Article Snippet: STAT3 small interfering RNA (sense: 5′-GGGACCUGGUGUGAAUUAUTT-3′, antisense: 5′-AUAAUUCACACCAGGUCCCTT-3′) and scrambled control siRNA (sense: 5′-UUCU CCGAACGUG UCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGA GAATT-3′) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Flow Cytometry, Staining, Isolation, Western Blot, Control

Journal: Oncotarget

Article Title: Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

doi: 10.18632/oncotarget.7051

Figure Lengend Snippet: ( A ) H292 cells were treated with 15 μM physalin A (PA) for 4 h. Immunofluorescence analysis was performed with a rabbit anti-p-STAT3 (Tyr 705) antibody followed by an anti-rabbit IgG Fab2 Alexa Fluor 555. Nuclei were stained with DAPI. Merge image showed the overlay of red Alexa Fluor 555 and blue DAPI fluorescence. ( B ) H292 cells were co-transfected with a STAT3-luciferase plasmid and a Renilla luciferase plasmid, and after 24 h, the cells were treated with 15 μM PA for 8 h. Cell lysates were analyzed for luciferase activity. Results are mean ± SD from three independent experiments. *, †, ‡ p < 0.05, significantly different from *untreated control cells and those treated with † 5 μM or ‡ 10 μM PA.

Article Snippet: STAT3 small interfering RNA (sense: 5′-GGGACCUGGUGUGAAUUAUTT-3′, antisense: 5′-AUAAUUCACACCAGGUCCCTT-3′) and scrambled control siRNA (sense: 5′-UUCU CCGAACGUG UCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGA GAATT-3′) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunofluorescence, Staining, Fluorescence, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Control

Journal: Oncotarget

Article Title: Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

doi: 10.18632/oncotarget.7051

Figure Lengend Snippet: ( A ) H292 cells were incubated with the indicated concentrations of physalin A for 4 h, and the phosphorylation levels of the Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) and Src were determined by Western blotting. ( B ) H1975 and H358 cells were incubated with 15 μM physalin A for 4 h, and the phosphorylation levels of Jak2 and Jak3 were determined by Western blotting. ( C ) H292 cells were incubated with 15 μM physalin A for 0.5, 1, 2 and 4 h, and Western blotting was performed to determine the time-dependent phosphorylation level of Jak1, Jak2, Jak3, Tyk2 and SRC and STAT3. β-actin was used as a loading control.

Article Snippet: STAT3 small interfering RNA (sense: 5′-GGGACCUGGUGUGAAUUAUTT-3′, antisense: 5′-AUAAUUCACACCAGGUCCCTT-3′) and scrambled control siRNA (sense: 5′-UUCU CCGAACGUG UCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGA GAATT-3′) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Incubation, Phospho-proteomics, Western Blot, Control

Journal: Oncotarget

Article Title: Physalin A exerts anti-tumor activity in non-small cell lung cancer cell lines by suppressing JAK/STAT3 signaling

doi: 10.18632/oncotarget.7051

Figure Lengend Snippet: When subcutaneous H292 tumor xenografts reached 100–200 mm3, mice received PBS (1% DMSO and 5% Tween80), 40 mg/kg physalin A, 80 mg/kg physalin A, or 5 mg/kg cisplatin via intraperitoneal injection for 10 days. ( A ) Tumor volumes were measured at the indicated times using Vernier calipers. ( B ) The average tumor weight and ( C ) body weight of each group was measured on the last day of the experiment (day 10). ( D ) The anti-tumor rate was also determined at the end of the experiment. ( E ) Protein levels of pSTAT3 (Tyr705), total STAT3 and cleaved-caspase 3 in tumor tissues of mice in each treatment group. β-actin was used for normalization. *, †, ‡ p < 0.05, significantly different from the *control, † PA (40 mg/kg), and ‡ PA (80 mg/kg) groups.

Article Snippet: STAT3 small interfering RNA (sense: 5′-GGGACCUGGUGUGAAUUAUTT-3′, antisense: 5′-AUAAUUCACACCAGGUCCCTT-3′) and scrambled control siRNA (sense: 5′-UUCU CCGAACGUG UCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGA GAATT-3′) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Injection, Control

Journal: The Journal of Biological Chemistry

Article Title: β-Cell mass restoration by α7 nicotinic acetylcholine receptor activation

doi: 10.1074/jbc.RA118.004617

Figure Lengend Snippet: STAT3 dependence of α7nAChR anti-inflammatory signaling in β-cells. A, β-cells express α7R. RT-PCR reveals Chrna7 expression (encoding α7R) in mouse islets (upper panel) corroborates anti-α7R immunoblot (IB, lower panel). B, time course of α7R-mediated STAT3 phosphorylation (Thr705) in INS-1 cells. INS-1 cells were treated with α7R agonist (PNU, 100 μm) in heat-inactivated serum medium. Cell lysates were collected at intervals of 10 min, and immunoblotting was performed for phosphorylated and total STAT3. Band densitometry demonstrates that α7R agonist led to progressive STAT3 phosphorylation, peaking at 60 min (*, p < 0.05). C, generation of INS-1 cells with shRNA-mediated stable KD of STAT3 protein and scrambled shRNA control (scr). INS-1 cells were transfected with either GFP-tagged shRNA or scrambled shRNA as a control, and GFP-positive INS-1 cells were obtained through puromycin selection. Effective STAT3 KD was determined by immunoblot assessing total STAT3 protein levels. STAT3 protein level in the selected KD clone was reduced 60% compared with the scrambled control. These are representative live cell fluorescence images of GFP-positive shRNA and scrambled control INS-1 cells. D, INS-1 cells exhibit STAT3-dependent activation of α7R, required for anti-inflammatory signaling. Representative immunoblots from INS-1 cells with STAT3 KD and control cells subjected to proinflammatory cytokine challenge (TNFα, IL-1β, and IFNγ) ± PNU treatment reveal reduced proinflammatory signaling through decreased phospho-NF-κB and increased phospho-STAT3 levels, resulting in reduced iNOS induction in scrambled control shRNA INS-1 cells, which was abrogated in INS-1 cells with STAT3 KD. Band intensity quantitation was performed from three separate blots (*, p < 0.05).

Article Snippet: Membranes were incubated overnight at 4 °C with one of the following antibodies: total STAT3 (rabbit monoclonal, Cell Signaling Technology), phospho-STAT3–Tyr 705 (rabbit monoclonal, Cell Signaling), phospho-NF-κB–Ser 536 (rabbit monoclonal, Cell Signaling), iNOS/NOS2 (rabbit polyclonal, Millipore), IRS-2 (rabbit polyclonal, Cell Signaling Technology), phospho-Akt–Ser 473 (rabbit monoclonal, Cell Signaling Technology), total Akt1 (rabbit monoclonal, Cell Signaling Technology), total CREB (rabbit polyclonal, Cell Signaling Technology), phospho-CREB–Ser 133 , and Pdx1 (rabbit polyclonal, Millipore).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, shRNA, Transfection, Selection, Fluorescence, Activation Assay, Quantitation Assay

Journal: The Journal of Biological Chemistry

Article Title: β-Cell mass restoration by α7 nicotinic acetylcholine receptor activation

doi: 10.1074/jbc.RA118.004617

Figure Lengend Snippet: Cultured islets display α7nAChR-specific anti-inflammatory signaling. A, α7R agonist (PNU) treatment increased p-STAT3 (Thr705) levels 2-fold compared with vehicle-treated islets. Freshly isolated B6N islets were equilibrated overnight in a standard islet culture medium followed by a 1-h incubation in heat-inactivated serum–supplemented medium prior to exposure to α7R agonist (100 μm PNU for 1 h) or vehicle (DMSO). The densitometry data represent ratios of p-STAT3/total STAT3 performed on three separate experiments (*, p < 0.05). B, PNU curtailed proinflammatory marker expression in mouse primary islets, whereas its antagonist (MLA) restored proinflammatory marker expression in response to proinflammatory cytokine challenge. Freshly isolated islets were rested overnight in islet culture medium followed by a 1-h incubation in heat-inactivated serum–supplemented medium prior to exposure to α7R agonist or antagonist pretreatment before cytokine challenge. Shown are representative immunoblots and corresponding quantitation of inflammatory markers of islets subjected to cytokines and treated with α7R agonist or antagonist. Islet α7R activation drives the anti-inflammatory response with reduced phospho-NF-κB and increased phospho-STAT3 levels resulting in decreased iNOS induction, whereas α7R antagonism reverses the proinflammatory signaling response by inhibiting STAT3 phosphorylation and restoring phospho-NF-κB levels leading to enhanced iNOS levels. C, islet anti-inflammatory signaling depends on Chrna7 expression. WT, Chrna7 haplodeficient (Het), and Chrna7 null (KO) islets underwent cytokine challenge as described under “Experimental procedures.” Representative iNOS immunoblots and corresponding quantitation reveal that α7R agonist reduction of cytokine-induced iNOS generation depends on at least one functional Chrna7 gene copy. Band intensity quantitation in panels was performed from three separate immunoblots (*, p < 0.05).

Article Snippet: Membranes were incubated overnight at 4 °C with one of the following antibodies: total STAT3 (rabbit monoclonal, Cell Signaling Technology), phospho-STAT3–Tyr 705 (rabbit monoclonal, Cell Signaling), phospho-NF-κB–Ser 536 (rabbit monoclonal, Cell Signaling), iNOS/NOS2 (rabbit polyclonal, Millipore), IRS-2 (rabbit polyclonal, Cell Signaling Technology), phospho-Akt–Ser 473 (rabbit monoclonal, Cell Signaling Technology), total Akt1 (rabbit monoclonal, Cell Signaling Technology), total CREB (rabbit polyclonal, Cell Signaling Technology), phospho-CREB–Ser 133 , and Pdx1 (rabbit polyclonal, Millipore).

Techniques: Cell Culture, Isolation, Incubation, Marker, Expressing, Western Blot, Quantitation Assay, Activation Assay, Functional Assay

Journal: iScience

Article Title: Role of Fyn and the interleukin-6-STAT-3-autophagy axis in sarcopenia

doi: 10.1016/j.isci.2023.107717

Figure Lengend Snippet: Muscular autophagy in the tail-suspension secondary sarcopenia model (A) Selective muscle sizes from control and TS mice (n = 6 per group). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. control. (B) mRNA expression levels of atrogin-1 and MuRF-1, relative to β-actin, in the gastrocnemius muscles of TS mice (n = 3). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. control. (C) Representative immunoblots from three independent experiments with gastrocnemius muscles of control and TS mice, developed using the indicated antibodies. Signal quantification of the protein expression levels of Fyn, normalized to GAPDH (n = 3). Data are shown as mean ± SEM. ∗∗∗p < 0.005 vs. control. Signal quantification of the expression levels of total STAT3, normalized to GAPDH, and phosphoY705-STAT3, normalized with total STAT3 (n = 3). Data are shown as mean ± SEM. ∗∗∗p < 0.005 vs. control. (D) Representative H&E staining of control and TS mice. See also Figure S2 . (E) Representative p62 immunofluorescence visualized in control and TS mice. Proportion of p62-positive myocytes in control and TS mice (n = 3). Data are expressed as mean ± SEM. ∗p < 0.05 vs. control. (F) Three-month-old control and TS mice were treated with either vehicle (as control) or 0.4 mg/kg/day colchicine for two days. Immunoblots of the TA muscle were obtained using the indicated antibodies. Representative immunoblots of three independent experiments. Signal quantification of the expression levels of LC3-II, normalized with GAPDH levels (n = 3). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. control. The Mann–Whitney U test was used for statistical comparison. The bars on each column show standard error of the mean. Scale bars in the tissue images represent the lengths defined in each figure. Abbreviations: TA, tibialis anterior; TS, tail suspension; SEM, standard error of the mean; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; STAT3, signal transducer and activator of transcription 3; C, control; Col, colchicine.

Article Snippet: Rabbit polyclonal antibodies against STAT3 (1:1000) , Cell Signaling , CS9132; RRID: AB_331588.

Techniques: Suspension, Control, Expressing, Muscles, Western Blot, Staining, Immunofluorescence, MANN-WHITNEY, Comparison

Journal: iScience

Article Title: Role of Fyn and the interleukin-6-STAT-3-autophagy axis in sarcopenia

doi: 10.1016/j.isci.2023.107717

Figure Lengend Snippet: Analysis of upstream activators of Fyn for STAT3 in C2C12 cells (A) Representative immunoblots from three independent experiments on lysates of C2C12 myoblasts and myotubes developed using the indicated antibodies. (B) C2C12 myotubes expressing either non-target or Fyn shRNA were developed using the indicated antibodies (a). Signal quantification of the expression levels of phosphoY705-STAT3 induced by IL-6 treatment, normalized to total STAT3 (n = 3). Data are expressed as mean ± SEM. ∗∗∗p < 0.001 vs. non-target (b). The Mann-Whitney U test was used for statistical comparison. (C) IL-6 dose dependency on phosphorylation of STAT3 in C2C12 myotubes expressing either non-target or Fyn shRNA. (D) Time dependency of IL-6 treatment on phosphorylation of STAT3 in C2C12 myotubes expressing either non-target or Fyn shRNA. The bars on each column show standard error of the mean. Abbreviations: STAT3, signal transducer and activator of transcription 3; IL-6, interleukin 6.

Article Snippet: Rabbit polyclonal antibodies against STAT3 (1:1000) , Cell Signaling , CS9132; RRID: AB_331588.

Techniques: Western Blot, Expressing, shRNA, MANN-WHITNEY, Comparison, Phospho-proteomics

Journal: iScience

Article Title: Role of Fyn and the interleukin-6-STAT-3-autophagy axis in sarcopenia

doi: 10.1016/j.isci.2023.107717

Figure Lengend Snippet: Fyn-dependent STAT3 regulation of autophagy (A) Representative immunoblots from three independent experiments. After 15 min of IL-6 treatment, lysates of C2C12 myotubes were immunoprecipitated with Fyn antibody and immunoblots were prepared using the indicated antibodies. (B) Representative immunoblots from three independent experiments. After 15 min of either IL-1β or IL-6 treatment, lysates of C2C12 myotubes were immunoprecipitated with total STAT3 antibody, followed by immunoblotting with Fyn. (C) Representative immunoblots from three independent experiments indicating autophagy flux in myotube lysates, determined after NH 4 Cl/leupeptin treatment for 2 h, followed by IL-6 treatment. Lysates were immunoblotted with the indicated antibodies (a). Signal quantification of the expression levels for LC3-II, normalized to GAPDH (n = 3) (b). Data are expressed as mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.005 vs. control. The Mann-Whitney U test was used for statistical comparison. The bars on each column show standard error of the mean. Abbreviations: STAT3, signal transducer and activator of transcription; IL, interleukin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SEM, standard error of the mean; N/L, NH 4 Cl/leupeptin.

Article Snippet: Rabbit polyclonal antibodies against STAT3 (1:1000) , Cell Signaling , CS9132; RRID: AB_331588.

Techniques: Western Blot, Immunoprecipitation, Expressing, Control, MANN-WHITNEY, Comparison

Journal: iScience

Article Title: Role of Fyn and the interleukin-6-STAT-3-autophagy axis in sarcopenia

doi: 10.1016/j.isci.2023.107717

Figure Lengend Snippet: Fyn–STAT3-dependent sarcopenia (A) Selective muscle sizes of WT and Fyn-KO mice in the control and TS groups (n = 6 per group). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. WT. (B) mRNA expression levels of atrogin-1 and MuRF-1, relative to β-actin, in the gastrocnemius muscles of WT and Fyn-KO mice with TS vs. controls (n = 3). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. WT. (C) Immunoblots of gastrocnemius muscles of WT and Fyn-KO mice (TS and control) were developed using the indicated antibodies. Signal quantification of the expression levels for phosphoY705–STAT3, normalized to total STAT3 (n = 3). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. WT. (D) mRNA expression levels of IL-6, relative to β-actin, in the gastrocnemius muscles of WT and Fyn-KO mice (TS or control) (n = 3). Data are expressed as mean ± SEM. ∗∗∗p < 0.005 vs. WT. The Mann–Whitney U test was used for statistical comparison. The bars on each column show standard error of the mean. Abbreviations: WT, wild-type; Fyn-KO, Fyn-knockout; SEM, standard error of the mean; STAT3, signal transducer and activator of transcription 3; IL-6, interleukin 6.

Article Snippet: Rabbit polyclonal antibodies against STAT3 (1:1000) , Cell Signaling , CS9132; RRID: AB_331588.

Techniques: Control, Expressing, Muscles, Western Blot, MANN-WHITNEY, Comparison, Knock-Out

Journal: iScience

Article Title: Role of Fyn and the interleukin-6-STAT-3-autophagy axis in sarcopenia

doi: 10.1016/j.isci.2023.107717

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal antibodies against STAT3 (1:1000) , Cell Signaling , CS9132; RRID: AB_331588.

Techniques: Bioprocessing, Recombinant, Protease Inhibitor, Bicinchoninic Acid Protein Assay, Lysis, cDNA Synthesis, shRNA, Software

Journal: Cancer Management and Research

Article Title: Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway

doi: 10.2147/CMAR.S228984

Figure Lengend Snippet: Effect of CPXM2 on the proliferation and metastasis of hepatic stellate cells in vitro. ( A ) Examination of the effect of CPXM2 on the phosphorylation levels of JAK2 and Stat1 in LX2 cells using Western blot analysis. ( B ) Relative protein expression level of CPXM2 and the phosphorylation status of JAK2 and Stat1 in LX2 cells. **P<0.01. ( C ) Examination of CPXM2 in LX2 cells using immunofluorescence. The CPXM2 protein was stained red (labeled with PE-conjugated secondary antibody), and the nucleus was stained blue (stained with DAPI). Scale bar, 20 µm. ( D ) Growth curves of LX2 cells transfected with a CPXM2 overexpression plasmid determined by Cell Counting Kit-8 assay. **P<0.01. ( E ) A plate colony formation assay was used to determine the effect of CPXM2 on the proliferative capacity of LX2 cells. **P<0.01. ( F ) Matrigel invasion assay was used to examine the effect of CPXM2 on the invasive capacity of LX2 cells in vitro (left), and corresponding statistical analysis of invasive cells (right). Scale bar, 20 µm; **P<0.01. ( G ) Wound-healing assay to detect the effect of CPXM2 on the migratory ability of LX2 cells in vitro (left), and the corresponding statistical analysis (right). **P<0.01. Abbreviations: OD, optical density; CPXM2, carboxypeptidase X, M14 family member 2; Stat1, signal transducer and activator of transcription 1; JAK2, Janus kinase 2.

Article Snippet: Then, the separated proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skimmed milk diluted in TBS-Tween 20 buffer (TBST) at room temperature (RT) for 2 h. Subsequently, the PVDF membranes were probed with primary antibodies against human gp130 (cat. no. 9145, Cell Signaling Technology, Inc), Stat1 (cat. no. 9139, Cell Signaling Technology, Inc), phospho- Stat1 (cat. no. 9167, Cell Signaling Technology, Inc), phospho-JAK2 (cat. no. 68790, Cell Signaling Technology, Inc), JAK2 (cat. no. 13531, Cell Signaling Technology, Inc), CPXM2 (cat. no. ab201077, Abcam, USA) or β-actin (cat. no. ab8226, Abcam, USA) at a dilution of 1:1000 at 4°C overnight.

Techniques: In Vitro, Phospho-proteomics, Western Blot, Expressing, Immunofluorescence, Staining, Labeling, Transfection, Over Expression, Plasmid Preparation, Cell Counting, Colony Assay, Invasion Assay, Wound Healing Assay

Journal: Cancer Management and Research

Article Title: Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway

doi: 10.2147/CMAR.S228984

Figure Lengend Snippet: The effects of shRNA-mediated silencing of CPXM2 expression in HCC cells. ( A ) Western blotting was used to examine the effects of CPXM2 silencing and the activation of the JAK2/Stat1 signaling pathway in Huh1 cells. ( B ) Corresponding statistical analysis of the activation of the JAK2/Stat1 signaling pathway. **P<0.01 vs scramble. ( C ) Growth curves of Huh1 cells determined using a Cell Counting Kit-8 assay. **P<0.01 vs scramble. ( D ) A colony formation assay was used to explore the proliferative capacity of Huh1 cells. **P<0.01 vs scramble. ( E ) A Matrigel invasion assay to examine the impact of CPXM2 silencing on the invasive ability of Huh1 cells in vitro, and corresponding statistical analysis of the number of invading cells. Scale bar, 20 µm. **P<0.01 vs scramble. ( F ) Wound-healing assay to determine the migratory ability of Huh1 cells in vitro (left), and the corresponding statistical analysis (right). **P<0.01 vs scramble. Abbreviations: shRNA, short hairpin RNA; HCC, hepatocellular carcinoma; CPXM2, carboxypeptidase X, M14 family member 2; Stat1, signal transducer and activator of transcription 1; JAK2, Janus kinase 2; p-, phosphorylated.

Article Snippet: Then, the separated proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skimmed milk diluted in TBS-Tween 20 buffer (TBST) at room temperature (RT) for 2 h. Subsequently, the PVDF membranes were probed with primary antibodies against human gp130 (cat. no. 9145, Cell Signaling Technology, Inc), Stat1 (cat. no. 9139, Cell Signaling Technology, Inc), phospho- Stat1 (cat. no. 9167, Cell Signaling Technology, Inc), phospho-JAK2 (cat. no. 68790, Cell Signaling Technology, Inc), JAK2 (cat. no. 13531, Cell Signaling Technology, Inc), CPXM2 (cat. no. ab201077, Abcam, USA) or β-actin (cat. no. ab8226, Abcam, USA) at a dilution of 1:1000 at 4°C overnight.

Techniques: shRNA, Expressing, Western Blot, Activation Assay, Cell Counting, Colony Assay, Invasion Assay, In Vitro, Wound Healing Assay

Journal: Cancer Management and Research

Article Title: Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway

doi: 10.2147/CMAR.S228984

Figure Lengend Snippet: shRNA was used to silence gp130 expression in CPXM2-expressing LX2 cells. ( A ) Western blotting was used to examine the effects of gp130 silencing on the phosphorylation status of JAK2 and Stat1 in CPXM2-expressing LX2 cells. **P<0.01 vs scramble. ( B ) Quantitative analysis of JAK2/Stat1 phosphorylation levels. **P<0.01 vs scramble. ( C ) Growth curves of LX2 cells determined using a Cell Counting Kit-8 assay. **P<0.01 vs scramble. ( D ) A colony formation assay was used to determine the proliferative capacity of LX2 cells with gp130 silenced. **P<0.01 vs scramble. ( E ) A Matrigel invasion assay was used to examine the impact of gp130 silencing on the invasiveness of LX2 cells in vitro (left), and corresponding statistical analysis of the invasiveness cells (right). Scale bar, 20 µm. **P<0.01 vs scramble. ( F ) A wound-healing assay was used to examine the migratory ability of the LX2 cell line in vitro (left), and corresponding statistical analysis (right). **P<0.01 vs scramble. Abbreviations: shRNA, short hairpin RNA; Stat1, signal transducer and activator of transcription 1; JAK2, Janus kinase 2; p-, phosphorylated; gp130, glycoprotein 130; CPXM2, carboxypeptidase X, M14 family member 2.

Article Snippet: Then, the separated proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skimmed milk diluted in TBS-Tween 20 buffer (TBST) at room temperature (RT) for 2 h. Subsequently, the PVDF membranes were probed with primary antibodies against human gp130 (cat. no. 9145, Cell Signaling Technology, Inc), Stat1 (cat. no. 9139, Cell Signaling Technology, Inc), phospho- Stat1 (cat. no. 9167, Cell Signaling Technology, Inc), phospho-JAK2 (cat. no. 68790, Cell Signaling Technology, Inc), JAK2 (cat. no. 13531, Cell Signaling Technology, Inc), CPXM2 (cat. no. ab201077, Abcam, USA) or β-actin (cat. no. ab8226, Abcam, USA) at a dilution of 1:1000 at 4°C overnight.

Techniques: shRNA, Expressing, Western Blot, Phospho-proteomics, Cell Counting, Colony Assay, Invasion Assay, In Vitro, Wound Healing Assay