stat5 silencing  (Santa Cruz Biotechnology)

Journal: Heliyon

Article Title: Insulin augments angiotensin II-induced myocardial fibrosis via the MEK/STAT3 pathway

doi: 10.1016/j.heliyon.2023.e22860

Figure Lengend Snippet: Insulin facilitated Ang II-mediated STAT expression and activation in rat heart tissue. Protein levels of p-STAT1, STAT1, p-STAT3, STAT3, p-STAT5 and STAT5 in the heart tissues of insulin-treated rats with or without Ang II-induced heart failure were detected by WB analysis, and the phosphorylation of STAT1, 3, and 5 was determined by calculating the ratio of p-STATs/STATs. The original image of Western blot is in the supplementary material. *, P < 0.05 compared with the control group; Data are expressed means ± SEM, determined in three independent experiments. Oneway ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze difference between the groups. ***, P < 0.001. #, P < 0.05 compared to the insulin group, ##, P < 0.01; ###, P < 0.001. &, P < 0.05 compared with the Ang II group, &&, P < 0.01; &&&, P < 0.001.

Article Snippet: STAT3 and STAT5 silencing was performed by transfection with small interfering RNAs (siRNAs) against STAT3 and STAT5 (sc-29494 and sc-29496; Santa Cruz Biotechnology, USA) as described in our previous study [ ].

Techniques: Expressing, Activation Assay, Phospho-proteomics, Western Blot, Control

Journal: Heliyon

Article Title: Insulin augments angiotensin II-induced myocardial fibrosis via the MEK/STAT3 pathway

doi: 10.1016/j.heliyon.2023.e22860

Figure Lengend Snippet: Insulin-mediated promotion of AngII-induced proliferation and collagen synthesis in rat CFs is dependent on the upregulation of STAT3. A, B: Primary rat cardiac fibroblasts were transfected with invalid, STAT5, or STAT3 siRNA for 24 h and were then exposed to 100 nmol/L Ang II and/or 100 nmol/L insulin for another 24 h. The protein levels of p-STAT3, STAT3 (A), p-STAT5 and STAT5 (B) were measured by WB analysis. C: A [3H]-thymidine incorporation assay was used to measure the proliferation of CFs. D: qPCR analysis of the mRNA expression of the fibrotic gene collagen COL1A1. The original image of Western blot is in the supplementary material. Data are expressed means ± SEM, determined in three independent experiments. Oneway ANOVA, followed by LSD t and SNK post hoc test, was performed to analyze difference between the groups. ***, P < 0.001, compared with the control group; ###compared with the Ang II group, P < 0.001.

Article Snippet: STAT3 and STAT5 silencing was performed by transfection with small interfering RNAs (siRNAs) against STAT3 and STAT5 (sc-29494 and sc-29496; Santa Cruz Biotechnology, USA) as described in our previous study [ ].

Techniques: Transfection, Thymidine Incorporation Assay, Expressing, Western Blot, Control

Journal: EMBO molecular medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia.

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: Figure 2. PRL‐3 protein expression decreases upon FLT3 or Src inhibition in AML cell lines. TF1-ITD and MOLM-14 cells were incubated with various concentrations of FLT3 inhibitors (PKC412, CEP-701) or Src inhibitors (SU6656, PP2) for 24 h. Whole cell lysates were subjected to Western blot analysis with indicated antibodies. GAPDH, loading control. A. (a–d) Western blot analysis of AML cells upon FLT3 inhibition. PKC412 and CEP-701 inhibited the phosphorylation of both FLT3 and STAT5 as well as PRL-3 protein levels in a dose-dependent manner in both TF1-ITD (a, b) and MOLM-14 (c, d) cells B. (a–d) Western blot analysis of AML cells upon FLT3 inhibition. PKC412 and CEP-701 inhibited the phosphorylation of Src, but not JAK, in a dose-dependent manner in both TF1-ITD (a, b) and MOLM-14 (c, d) cells. C. (a, b) Western blot analysis of AML cells upon Src inhibition. SU6656 (a) and PP2 (b) inhibited the phosphorylation of Src and STAT5 as well as PRL-3 protein levels in a dose-dependent manner in TF1-ITD cells.

Article Snippet: Transient transfection of siRNA or reporter vector TF‐1, TF1‐ITD, MOLM‐14 or MV4‐11 cells were re‐suspended at 2 106 cells per 100 ml of appropriate Nucleofector kit solution (Amaxa Biosystems, Cologne, Germany) and were nucleofected with 2 mg of FLT3 SMARTpool siRNA duplexes (Dharmacon Research, Milipore), PRL‐3 siRNA, Signal Silence STAT5 siRNA I/II (Cell Signalling Technologies), AP‐1 SEAP reporter vector, ERK siRNA, JNK siRNA or non‐ silencing siRNA (Santa Cruz Biotechnology, CA).

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

Journal: EMBO molecular medicine

Article Title: Oncogenic roles of PRL-3 in FLT3-ITD induced acute myeloid leukaemia.

doi: 10.1002/emmm.201202183

Figure Lengend Snippet: Figure 3. STAT5A is a direct transcriptional regulator of PRL‐3 expression. A. Two putative STAT5 binding sites (S1 and S2; DNA sequences illustrated) in a distal 50-flanking region of PRL-3, as predicted by TRANSFAC. B. EMSA analysis using S1 and S2 biotinylated DNA probes (S1 and S2) incubated with nuclear extracts from either TF-1 or TF1-ITD cells. Arrow, shifted protein/ probe complex. C. EMSA analysis as in (B) in the presence of 10-fold molar excess of unlabelled STAT5 competitor. D. Western blot analysis of streptavidin–agarose pull-down fractions (unbound or bound) using probe S1. E. Left panel: schematic diagram of a 5.4 kb upstream sequence of PRL-3 and its 50-sequential deletion sequence with luciferase reporter vector (pGL3-S1a, S1b, S1c and S1d), respectively. Right panel: STAT5A or STAT5B expression vectors were co-transfected with PRL-3 luciferase reporter vector to TF-1 cells and luciferase activity measured. Error bars represent the mean SD from three independent experiments. F. PRL-3 expression is down-regulated upon siRNA-mediated STAT5 depletion in AML cells. NS, control non-silencing siRNA. (a) Quantitative real time PCR analysis of PRL-3 mRNA level after knock-down of STAT5 gene, normalized to GAPDH mRNA. Statistical differences between two groups were determined using Student’s t-test (mean SD, n ¼ 3). p < 0.001 (for TF1-ITD); p ¼ 0.011 (for MOLM-14); p ¼ 0.038 (for MV4-11). (b) Western blot analysis of PRL-3 protein level after knock-down of STAT5 gene. GAPDH, loading control.

Article Snippet: Transient transfection of siRNA or reporter vector TF‐1, TF1‐ITD, MOLM‐14 or MV4‐11 cells were re‐suspended at 2 106 cells per 100 ml of appropriate Nucleofector kit solution (Amaxa Biosystems, Cologne, Germany) and were nucleofected with 2 mg of FLT3 SMARTpool siRNA duplexes (Dharmacon Research, Milipore), PRL‐3 siRNA, Signal Silence STAT5 siRNA I/II (Cell Signalling Technologies), AP‐1 SEAP reporter vector, ERK siRNA, JNK siRNA or non‐ silencing siRNA (Santa Cruz Biotechnology, CA).

Techniques: Expressing, Binding Assay, Incubation, Western Blot, Sequencing, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Control, Real-time Polymerase Chain Reaction, Knockdown