Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

doi: 10.33594/000000010

Figure Lengend Snippet: Fig. 1. STAT5B is a transcriptional regulator of Nos2. A) Chromosome ideogram of mouse Stat5b and Nos2 on chromosome 11(http://genome.ucsc.edu/). B) Cartoon representation of the putative binding site of STAT5B on Nos2 promoter, as identified from ChampionChiP Transcription Factor Search Portal (SABiosciences). C) Chromatin immunoprecipitation in MIN6 cells identified Nos2 as a direct target of STAT5B. Lane 1 - Input DNA, lane 2 - mouse control IgG, lane 3 - anti-RNA polymerase II antibody, lane 4 - anti-STAT5B antibody, lane 5 - DNA ladder. The image is a representation of experiment done thrice independently. D) Bar graph representation of 2-ΔΔCt of Stat5b and Nos2 transcripts showing downregulation in prolactin-induced Stat5b siRNA treated versus control siRNA treated MIN6 cells, calculated with respect to untreated cells and 18SrRNA as the endogenous control. (n = 3). E) Western blots and F) densitometric analysis of STAT5B and NOS2, showing a downregulation in PRL-induced siRNA treated samples, with actin as the loading control (n = 3). P-value was calculated using Student’s t-test.

Article Snippet: Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Nos2 via STAT5B Binding to Nos2 Gene Promoter Mediates Nitric Oxide Production: Relevance in β-Cell Maintenance.

doi: 10.33594/000000010

Figure Lengend Snippet: Fig. 4. Impact of Stat5b silencing on SOD2 and H2O2. A) Bar graph representation of increased relative transcript levels (2-

Article Snippet: Control siRNA (sc-37007) and Stat5b siRNA (sc-37011) were from Santa Cruz Biotechnology (SCBT), Dallas, TX, USA.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

doi: 10.3389/fphar.2018.00531

Figure Lengend Snippet: Icariin downregulates the constitutive STAT3, JAK1/2, and Src in U266 cells. (A) Chemical structure of icariin. (B) U266 cells (1 × 10 6 cells/well) were treated with the indicated concentrations of icariin for 8 h and different time periods with 100 μM. Then the same amounts of whole cell lysates were prepared and compared to assess inhibition for phospho-STAT3(Tyr705), and STAT3 by western blot analysis. (C) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations and different time periods. Thereafter nuclear extracts were studied for STAT3 inhibition levels by EMSA. (D) U266 cells (2 × 10 4 cells/well) were treated with 100 μM of icariin for 6 h, and analyzed distribution of STAT3 into the nucleus by immunocytochemistry. Datas were quantified on graph ( right panel ). (E) Specificity of STAT3 was confirmed by competition assay in U266 cells. Nuclear extract of U266 cells were binding with STAT3 consensus oligonucleotide ( second lane ) or mutant oligonucleotide ( third lane ). (F) U266 cells (1 × 10 6 cells/well) were treated with various indicated concentrations of icariin and different time periods. Then the expression of p-JAK1(Tyr1022/1023), JAK1, p-JAK2(Tyr1007/1008), JAK2, p-Src(Tyr416), and Src were analyzed by western blotting. The results shown are representative of three independent experiments.

Article Snippet: Nuclear extract was prepared using 30 × unlabeled STAT3 consensus oligonucleotide (GATCCTTCTCGGGAATTCCTAGATC-3′; BIONEER, Daejeon, Korea) or mutant STAT3 oligonucleotide (GATCCTTCTGGGCCGTCCTAGATC-3′ BIONEER, Daejeon, Korea) incubation for 20 min in room temperature.

Techniques: Inhibition, Western Blot, Immunocytochemistry, Competitive Binding Assay, Binding Assay, Mutagenesis, Expressing

Journal: Frontiers in Pharmacology

Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

doi: 10.3389/fphar.2018.00531

Figure Lengend Snippet: Icariin can inhibit inducible STAT3 in MM.1S cells. (A) MM.1S cells (1 × 10 6 cells/well) were treated with IL-6 (10 ng/ml) for different time periods. Then whole cell lysates were compared for p-STAT3(Tyr705) and STAT3 expression by western blot analysis to determine the optimal periods of p-STAT3 signaling. (B–D) MM.1S cells (1 × 10 6 cells/well) were treated with 100 μM icariin for different time periods and then stimulated with IL-6 (10 ng/ml) for 10 min. Equal amounts of whole cell lysates were prepared and expression of p-STAT3(Tyr705), STAT3, p-JAK(Tyr1022/1023), JAK1, p-JAK(Tyr1007/1008), JAK2, p-Src(Tyr416), and Src were analyzed by western blotting. (E) STAT3 promoter luciferase assay in MM.1S cells (2 × 10 6 cells/well) transfected for 48 h, and were treated with icariin (0, 10, 25, 50, 100 μM) for 8 h. Then stimulated with IL-6 (10 ng/ml) for 10 min. The results shown are representative of three independent experiments.

Article Snippet: Nuclear extract was prepared using 30 × unlabeled STAT3 consensus oligonucleotide (GATCCTTCTCGGGAATTCCTAGATC-3′; BIONEER, Daejeon, Korea) or mutant STAT3 oligonucleotide (GATCCTTCTGGGCCGTCCTAGATC-3′ BIONEER, Daejeon, Korea) incubation for 20 min in room temperature.

Techniques: Expressing, Western Blot, Luciferase, Transfection

Journal: Frontiers in Pharmacology

Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

doi: 10.3389/fphar.2018.00531

Figure Lengend Snippet: Icariin enhance Bortezomib in U266 cells. (A) To confirm the synergic effect on cytotoxicity of icariin and bortezomib (Bor), we used the MTT assay. U266 cells (1 × 10 4 cells/well) were incubated with icariin (0, 10, 25, and 50 μM) and bortezomib (0, 1, 2.5, and 5 nM) for 24 h. The average of CI values for various combinations shows that icariin increase cytotoxicity of bortezomib. The best combination ratio is 10 μM icariin and 1 nM bortezomib. (B) Live and Dead assay was performed to confirm the synergic effects on U266 cells (1 × 10 6 cells/well) with 10 μM icariin and 1 nM bortezomib for 24 h. Live cells were stained in green and dead cells were stained in red. The graph ( right) shows the rate of dead cells by quantification. (C,D) U266 cells (1 × 10 6 cells/well) were treated with 10 μM icariin and 1 nM bortezomib for 24 h. Then equal amounts of whole cell lysates were prepared and expression of p-STAT3(Tyr705), STAT3, p-JAK1(Tyr1022/1023), JAK1, p-JAK2(Tyr1007/1008), JAK2, p-Src(Tyr416), and Src were analyzed by western blotting. The results shown are representative of three independent experiments.

Article Snippet: Nuclear extract was prepared using 30 × unlabeled STAT3 consensus oligonucleotide (GATCCTTCTCGGGAATTCCTAGATC-3′; BIONEER, Daejeon, Korea) or mutant STAT3 oligonucleotide (GATCCTTCTGGGCCGTCCTAGATC-3′ BIONEER, Daejeon, Korea) incubation for 20 min in room temperature.

Techniques: MTT Assay, Incubation, Staining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

doi: 10.3389/fphar.2018.00531

Figure Lengend Snippet: Icariin and Bortezomib induce apoptosis by caspase-3 and PARP in U266 cells. (A) To confirm the synergistic effect between icariin and bortezomib on cell cycle, U266 cells (1 × 10 6 cells/well) were incubated with icariin (10 μM) and bortezomib (1 nM) for 24h then treated with RNase A for 1h. After staining with propidium iodide, cells were analyzed by flow cytometry. (B) U266 cells were treated with icariin and bortezomib for 24 h. Cells were fixed and stained with TUNEL assay reagent, then analyzed with a flow cytometer. (C,D) We confirm the synergistic effect for induced apoptosis by western blot analysis. U266 cells (1 × 10 6 cells/well) were treated with 10 μM icariin and 1 nM bortezomib for 24 h. Same amounts of whole cell lysates were prepared and probed using Bcl-2, Bcl-xl, Survivin, IAP-1, IAP-2, COX-2, VEGF, MMP-9, caspase-3, and PARP antibodies then analyzed by Western blotting. (E) To confirm the anti-cancer effect of icariin and bortezomib, U266 cells (1 × 10 6 cells/well) were incubated with icariin (10 μM) and bortezomib (1 nM) for 24h then proteins were resolved on SDS–PAGE and probed against p21 antibody. (F) U266 cells were transfected with STAT3 siRNA or scramble siRNA for 48 h, then 100 μM of icariin were treated for 24 h. The Whole cell lysates were prepared and 15 μg proteins were resolved on SDS–PAGE and probed against PARP antibody. b-actin was used as internal controls. (G) U266 cells were transfected with STAT3 siRNA or scramble siRNA for 48 h, then 100 μM of icariin were treated for 24 h. Then cell viability was analyzed by MTT assay. The results shown are representative of three independent experiments.

Article Snippet: Nuclear extract was prepared using 30 × unlabeled STAT3 consensus oligonucleotide (GATCCTTCTCGGGAATTCCTAGATC-3′; BIONEER, Daejeon, Korea) or mutant STAT3 oligonucleotide (GATCCTTCTGGGCCGTCCTAGATC-3′ BIONEER, Daejeon, Korea) incubation for 20 min in room temperature.

Techniques: Incubation, Staining, Flow Cytometry, TUNEL Assay, Western Blot, SDS Page, Transfection, MTT Assay

Journal: Frontiers in Pharmacology

Article Title: Anti-myeloma Effects of Icariin Are Mediated Through the Attenuation of JAK/STAT3-Dependent Signaling Cascade

doi: 10.3389/fphar.2018.00531

Figure Lengend Snippet: A schematic diagram showing the effects of icariin on STAT3 signaling pathways and apoptosis in MM cells.

Article Snippet: Nuclear extract was prepared using 30 × unlabeled STAT3 consensus oligonucleotide (GATCCTTCTCGGGAATTCCTAGATC-3′; BIONEER, Daejeon, Korea) or mutant STAT3 oligonucleotide (GATCCTTCTGGGCCGTCCTAGATC-3′ BIONEER, Daejeon, Korea) incubation for 20 min in room temperature.

Techniques: Protein-Protein interactions