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

Article Title: Periodic mechanical stress activates integrinβ1-dependent Src-dependent PLCγ1-independent Rac1 mitogenic signal in rat chondrocytes through ERK1/2.

doi: 10.1159/000341461

Figure Lengend Snippet: Fig. 6. Effects of Src on the expression, phosphorylation and activation of Rac1 under conditions of periodic mechanical stress. Chondrocytes were transfected with shRNA targeted to Src or with nontargeting NT sequences prior to lysis and Western blotting for Src protein. The Src shRNA sequence achieved about 50% reduction in Src protein level. After pretreatment with DMSO or Src inhibitor PP2, or Control shRNA or Src shRNA, rat chondrocytes were cultured in vitro for 1 h under static conditions or with periodic mechanical stress. The expression and phosphorylation levels of Rac1 were detected by western blotting, and Rac1 activation was quantiϐied by Rac1 GTPase activity assay. The total quantity of Rac1 served as the control. Results are represented in the histogram (n=5, *, p<0.05 for each). The images above are representative results of western blotting and Rac1 GTPase activity assay. The phosphorylation levels of Rac1-Ser71 and Rac1 activation levels in the PP2 and Src shRNA pretreatment groups were signiϐicantly diminished relative to those of the control groups in response to periodic mechanical stress (n=5, p<0.05 for each, Student unpaired t-test).

Article Snippet: Src shRNA (r) Lentiviral Particles, Control shRNA Lentiviral Particles and Polybrene were supplied by Santa Cruz (USA).

Techniques: Expressing, Phospho-proteomics, Activation Assay, Transfection, shRNA, Lysis, Western Blot, Sequencing, Control, Cell Culture, In Vitro, Activity Assay

Journal: Oncogene

Article Title: Dual-faced SH3BGRL: oncogenic in mice, tumor suppressive in humans

doi: 10.1038/onc.2015.391

Figure Lengend Snippet: Knockdown of c-Src abrogates mSH3BGRL-induced metastasis in nude mice. ( a ) Four independent pools of CT-26 SH3BGRL cells stably expressing c-Src-specific shRNA (SH3BGRL-SrcKD 1–4) were analyzed for the expression of c-Src compared with a scrambled control (SH3BGRL-scr). glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. ( b ) Lysates from exponentially growing stable cells (SH3BGRL-SrcKD is a combined pool from all four clones in ( a )) were immunoblotted with various antibodies. GAPDH served as a loading control. The averaged relative protein expression level in ( a ) and ( b ) is quantified and shown under the immediate panels. ( c ) In total, 1 × 10 6 CT-26 SH3BGRL or CT-26 SH3BGRL-SrcKD cells were injected intravenously into the tail vein of nude mice. After 14 days, mice were killed and their lungs were photographed and scored for metastatic tumor nodules (mean±s.d., n =4, *** P <0.001).

Article Snippet: For knockdown of c-Src in CT-26 cells overexpressing SH3BGRL, four shRNA constructs against mouse Src (OriGene, Rockville, MD, USA; cat. no. TG514248) were transfected to generate stable cell pools using 1 μg/ml puromycin selection for 3–4 weeks.

Techniques: Stable Transfection, Expressing, shRNA, Clone Assay, Injection

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 through GHR signaling (A) Western blots of hNCC line #1 pretreated with 20 mg/mL pegvisomant (Peg) for 1 h then treated with GH (500 ng/mL) for an additional 24 h. C, untreated control. (B) Colon tissue derived from 3- and 24-month-old WT and GHR −/− male mice. Each lane represents sample analysis derived from an individual animal. (C) hNCC line #1 transduced with lentivirus expressing GH (Lenti-GH) or empty vector (Lenti-V) and analyzed 10 days later. (D) iPSC-derived human intestinal organoid lines from 3 different patients were transduced with Lenti-V or Lenti-GH and analyzed 5 weeks later. Representative blots from one human intestinal organoid line are shown. (E) hNCC line #1 transduced with lentivirus expressing control shRNA (shControl) or GH shRNA (shGH). (F) Three different human intestinal organoid lines were transduced with GFP-expressing Lenti-V or Lenti-GH and cultured for 5 weeks, then sorted for GFP-negative cells. GFP-negative cells co-existing in organoids with either Lenti-GH or Lenti-V expressing cells were analyzed. All lines exhibited similar results. Representative blots from one human intestinal organoid line are shown. Representative blots from at least 3 independent experiments are depicted. ImageJ quantifications of western blots are depicted in Figure S2 .

Article Snippet: Lentiviral particles expressing human GH shRNA, Src shRNA or nontargeted control shRNA (GFP Control Lentiviral Particles) are from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Derivative Assay, Transduction, Expressing, Plasmid Preparation, shRNA, Cell Culture

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 by activating Src/AMPK (A and B) Western blots of (A) hNCC line #1 treated with 1 μM AMPK inhibitor (Compound C) for 1 h followed by GH (500 ng/mL) for an additional 24 h and (B) human intestinal organoids treated with 2 μM AMPK inhibitor and GH (500 ng/mL) for 48 h. (C–F) Western blots of hNCC line #1 was treated with (C) GH (500 ng/mL) for up to 60 min; (D) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 30 min–60 min; (E) 25 μM Src inhibitor for 1 h followed by GH (500 ng/mL) for 24 h; and (F) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 3 h. Untreated cells/organoids served as control (C). (G–I) Western blots of hNCC line #1 transduced with lentivirus expressing Control shRNA (shControl) or Src shRNA (shSrc) (G) analyzed 72 h after transduction; (H) treated with GH (500 ng/mL) for 3 h; and (I) treated with GH (500 ng/mL) for 24 h. ImageJ quantifications of western blots are depicted in Figure S5 .

Article Snippet: Lentiviral particles expressing human GH shRNA, Src shRNA or nontargeted control shRNA (GFP Control Lentiviral Particles) are from Santa Cruz Biotechnology.

Techniques: Western Blot, Control, Transduction, Expressing, shRNA

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet:

Article Snippet: Lentiviral particles expressing human GH shRNA, Src shRNA or nontargeted control shRNA (GFP Control Lentiviral Particles) are from Santa Cruz Biotechnology.

Techniques: Virus, Control, shRNA, Recombinant, Cell Recovery, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, DC Protein Assay, Extraction, Immunoprecipitation, cDNA Synthesis, Single Cell Gel Electrophoresis, Derivative Assay, Generated, Software, Imaging, Real-time Polymerase Chain Reaction, Microscopy

Journal: Hepatology (Baltimore, Md.)

Article Title: Molecular subtype and response to dasatinib, an Src/Abl small molecule kinase inhibitor, in hepatocellular carcinoma cell lines in vitro.

doi: 10.1002/hep.26223

Figure Lengend Snippet: Fig. 4. Src knockdown using lentivirus shRNA gene transduction. (A) An effective decrease in total src and phospho-src in two cell lines that are sensitive to dasatinib. In both HLE and SNU 423 cells, there is a significant decrease in the transfected clones (SRC7) as com- pared to the vector control cells (C7). A431 lysates were used as a positive control (þ). (B) The results of cell count assays between the vector control and src knockdowns for both lines. As can be seen, de- spite knockdown of src and activated src, there are no effects on cell number over time.

Article Snippet: Lentiviral particles containing c-SRC shRNA (Santa Cruz Biotechnology) was diluted with transduction media consisting of serum-free, antibiotic-free media and 5 lg/mL of Polybrene (Santa Cruz Biotechnology) to a multiplicity of infection (MOI) 7 and 13.

Techniques: Knockdown, shRNA, Transduction, Transfection, Clone Assay, Plasmid Preparation, Control, Positive Control, Cell Counting

Journal: Biochimica et biophysica acta

Article Title: Estradiol increases cell growth in human astrocytoma cell lines through ERα activation and its interaction with SRC-1 and SRC-3 coactivators.

doi: 10.1016/j.bbamcr.2011.11.004

Figure Lengend Snippet: Fig. 4. Effect of SRC-1 and SRC-3 silencing on changes in cell number induced by PPT in U373 and D54 human astrocytoma cell lines. U373 (Left panel) and D54 (Right panel) cell lines were transfected with SRC-1 shRNA, SRC-3 siRNA and control RNAs interference, and then treated with PPT (1 nM). Every day during six days cells were harvested and cell number was determined as in Fig. 1. Representative Western blots of SRC-1 and SRC-3 from cells lysed at day 3 after PPT treatment are shown in each panel. Mock: vehicle control (cells treated with transfection reagent without RNA interference). Data are mean±S.E.M. n=4. *Pb0.05 vs. Mock, Control shRNA and Control siRNA. **Pb0.05 vs. Mock.

Article Snippet: Four different RNA interferences were purchased from Santa Cruz Biotechnology (CA, USA): one short hairpin RNA (shRNA) targeting coactivator SRC-1, one small interfering RNA (siRNA) targeting coactivator SRC-3 and two control RNAs, shRNA and siRNA, respectively.

Techniques: Transfection, shRNA, Control, Western Blot

Journal: Experimental and Therapeutic Medicine

Article Title: Dual inhibition of MET and SRC kinase activity as a combined targeting strategy for colon cancer

doi: 10.3892/etm.2017.4692

Figure Lengend Snippet: Effect of EGF and IGF-1 on MET activation. EGF (25 ng/ml) was added to (A) HT-29 and (B) HCT-116 cells and the expression and phosphorylation of EGFR, MET, SRC, AKT and ERK was assessed by western blotting. IGF-1 (25 ng/ml) was added to (C) HT-29 and (D) HCT-116 cells. The expression and phosphorylation of IGF-1R, MET, SRC, AKT and ERK were observed by western blotting. HCT-116 cells were transiently transfected with scramble control or MET siRNA, and then 25 ng/ml (E) EGF or (F) IGF-1 was added for 2 h. The expression and phosphorylation of MET and SRC were studied by western blotting. Actin was used as a loading control. EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; siRNA, small interfering RNA; p, phosphorylated.

Article Snippet: The MET and SRC siRNA (5 nM) sequences from Shanghai GenePharma Co., Ltd. (Shanghai, China) were as follows: 5′-GCCUGAAUGAUGACAUUCU-3′ and 5′-GGCUCCAGAUUGUCAACAAtt-3′, respectively.

Techniques: Activation Assay, Expressing, Phospho-proteomics, Western Blot, Transfection, Control, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: Dual inhibition of MET and SRC kinase activity as a combined targeting strategy for colon cancer

doi: 10.3892/etm.2017.4692

Figure Lengend Snippet: MET inhibition by siRNA or inhibitor. (A) HT-29 and HCT-116 cells were transiently transfected with scramble control or MET siRNA for 48 h in 96-well plates. Cell viability was assessed by MTT assay. (B) Increasing concentrations of PHA-665752 (0.2, 1.0 and 5.0 µM) were added to HT-29 and HCT-116 cells, and cell viability was assessed after 48 h. Data are presented as the mean ± standard deviation of three independent experiments. Increasing concentrations of PHA-665752 (0.2, 1.0 and 5.0 µM) were added to (C) HT-29 and (D) HCT-116 cells for 24 h. The expression and phosphorylation of MET, SRC, AKT and ERK were studied by western blotting. (E) HCT-116 cells were pretreated with 0.2 µM PHA-665752 for 24 h after which they were stimulated with 25 ng/ml HGF for 2 h. The expression and phosphorylation of MET, SRC, AKT and ERK were analyzed by western blotting. Actin was used as a loading control. *P<0.05 vs. scramble control. siRNA, small interfering RNA; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; p, phosphorylated.

Article Snippet: The MET and SRC siRNA (5 nM) sequences from Shanghai GenePharma Co., Ltd. (Shanghai, China) were as follows: 5′-GCCUGAAUGAUGACAUUCU-3′ and 5′-GGCUCCAGAUUGUCAACAAtt-3′, respectively.

Techniques: Inhibition, Transfection, Control, MTT Assay, Standard Deviation, Expressing, Phospho-proteomics, Western Blot, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: Dual inhibition of MET and SRC kinase activity as a combined targeting strategy for colon cancer

doi: 10.3892/etm.2017.4692

Figure Lengend Snippet: Regulation of SRC on MET and non-ligand mediated MET activation. (A) Whole-cell extracts from HCT-116 cells were immunoprecipitated with anti-MET antibody. The immunoprecipitates were probed with MET and SRC antibodies. The input represents cell lysates that were not subjected to immunoprecipitation. Control immunoprecipitation was performed using IgG M. HCT-116 cells were transiently transfected with scramble control and (B) SRC siRNA for 48 h or (C) flag-tagged wild type SRC plasmid for 24 h. The expression of SRC, MET and p-MET was studied by western blotting. Increasing concentrations of dasatinib (0.1, 0.5 and 2.5 µM) were added to (D) HT-29 and (E) HCT-116 cells, and the expression and phosphorylation of SRC, MET, AKT and ERK was analyzed by western blotting. (F) HT-29 cells were pretreated with 0.1 µM dasatinib for 24 h, then stimulated with 25 ng/ml HGF for 2 h. The expression and phosphorylation of MET, SRC, AKT and ERK was examined by western blotting. HT-29 cells were pretreated with 0.1 µM dasatinib for 24 h, then stimulated with 25 ng/ml (G) EGF or (H) IGF-1 for 2 h. The expression and phosphorylation of EGFR, IGF-1R, MET and SRC was studied by western blotting. Actin was used as a loading control. IgG M, immunoglobulin G mouse; siRNA, small interfering RNA; p, phosphorylated; AKT, protein kinase B; ERK, extracellular signal-regulated kinase; HGF, hepatocyte growth factor; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; IGF-1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor.

Article Snippet: The MET and SRC siRNA (5 nM) sequences from Shanghai GenePharma Co., Ltd. (Shanghai, China) were as follows: 5′-GCCUGAAUGAUGACAUUCU-3′ and 5′-GGCUCCAGAUUGUCAACAAtt-3′, respectively.

Techniques: Activation Assay, Immunoprecipitation, Control, Transfection, Plasmid Preparation, Expressing, Western Blot, Phospho-proteomics, Small Interfering RNA