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: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

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: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

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: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

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: Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas

Article Title: Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1.

doi: 10.1590/s0100-879x2011007500150

Figure Lengend Snippet: Figure 4. Src shRNA and ERK1/2 shRNA inhibit periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. A, Chondrocytes were transfected with shRNA targeted to Src or ERK1/2 or with non-targeting (NT) sequences prior to lysis and Western blot for Src and ERK1/2 protein. Either the Src or ERK1/2 shRNA sequence achieved about a 50% reduction in Src or ERK1/2 protein levels. After pretreatment with control shRNA or Src shRNA and ERK1/2 shRNA, rat chondrocytes were cultured for 3 days under static conditions or conditions of periodic mechanical stress 8 h per day prior to proliferation studies. Cell proliferation was analyzed by direct cell counting (B) and CCK-8 assay (C). Rat chondrocytes were cultured in vitro for 8 h with static conditions or periodic mechanical stress. Aggrecan (D) and type II collagen (E) gene expression were measured by quantitative real-time PCR. Cell proliferation and matrix synthesis in the inhibitor pretreatment groups was significantly decreased relative to control in chondrocytes in response to periodic mechanical stress (N = 5, *P < 0.05, Student unpaired t-test).

Article Snippet: Src shRNA (r) Lentiviral Particles, ERK1 shRNA (r) Lentiviral Particles, ERK2 shRNA (r) Lentiviral Particles, Control shRNA Lentiviral Particles, and Polybrene were supplied by Santa Cruz.

Techniques: shRNA, Transfection, Lysis, Western Blot, Sequencing, Control, Cell Culture, Cell Counting, CCK-8 Assay, In Vitro, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas

Article Title: Periodic mechanical stress activates MEK1/2-ERK1/2 mitogenic signals in rat chondrocytes through Src and PLCγ1.

doi: 10.1590/s0100-879x2011007500150

Figure Lengend Snippet: Figure 6. Effect of PP2 and Src shRNA on the expression and phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 under con ditions of periodic mechanical stress. After pretreatment with DMSO or Src inhibitor (PP2), or Src shRNA or non-targeting (NT) sequence, rat chondrocytes were cultured in vitro for 1 h under static conditions or conditions of period ic mechanical stress. The expres sion and phosphorylation levels of Src (A), PLCγ1 (B), MEK1/2 (C), and ERK1/2 (D) were detected by Western blotting. For each West ern blot, the corresponding total amount of each signaling protein served as a control. The images above the histogram are repre sentative results of several inde pendent Western blots. The phos phorylation levels of Src-Tyr418 in the PP2 groups were significantly reduced compared to those in the control groups in chondrocytes in response to periodic mechanical stress (N = 5, *P < 0.05, Student unpaired t-test). The phospho rylation levels of PLCγ1-Tyr783, MEK-Ser217/221 and ERK1/2- Thr202/Tyr204 in the PP2 and Src shRNA groups were significantly reduced compared to those in the control groups in chondrocytes in response to periodic mechanical stress (N = 5, *P < 0.05, Student unpaired t-test).

Article Snippet: Src shRNA (r) Lentiviral Particles, ERK1 shRNA (r) Lentiviral Particles, ERK2 shRNA (r) Lentiviral Particles, Control shRNA Lentiviral Particles, and Polybrene were supplied by Santa Cruz.

Techniques: shRNA, Expressing, Phospho-proteomics, Sequencing, Cell Culture, In Vitro, Western Blot, Control

Journal: Journal of Bio-X Research

Article Title: GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

doi: 10.1097/jbr.0000000000000010

Figure Lengend Snippet: Figure 3. GPER1, but not ERa/b, is responsible for FAK phosphorylation in breast cancer cells. MCF-7 cells were exposed to either 10nM 17b-estradiol (E2) or 1m MGPER1 agonist G1 for 20minutes in the presence or absence of the pure ER antagonist ICI 182780 (ICI; 1mM) or GPER1 antagonist G15 (1mM). Top, Western blot of FAK and phosphorylated FAK; bottom, quantification of p-FAK. p-FAK densitometry values were adjusted to FAK intensity and then normalized to expression from the control sample. ∗P<.05, ∗∗P<.01, versus control; ###P<.001, versus E2 or G1. (B) MDA-MB-231 cells were transfected with empty vector or GPER1 plasmid for 48hours and then exposed to 10nM E2 or 1mM G1 for 20minutes. Western blot for p-FAK, FAK, GPER1and b-actin was performed. (C) MCF-7 cells were transfected with scrambled siRNA, ERa, ERb, or GPER1 targeted siRNA for 48hours. ERa, ERb, or GPER1protein expressions were detected by western blot; b-actin intensity was used as loading control. (D–F) MCF-7 cells were transfected with 100nM scrambled siRNA or target siRNAs for ERa, ERb, or GPER1 for 48hours and then treated with E2 (10nM) or G1 (1mM) for 20minutes. Top, Western blot of FAK or phosphorylated FAK; bottom, densitometric analysis of p-FAK. ∗P<.05, ∗∗P<.01, versus control; ##P<.01, versus E2. All the experiments were repeated at least three times with consistent results and representative images are shown. ER=estrogen receptor, GPER1=G-protein-coupled ER, FAK=focal adhesion kinase.

Article Snippet: The siRNAs used for experiments included ERa siRNA (#sc29305, Santa Cruz, CA), ERb siRNA (#SR301462, OriGene, Rockville, MD, USA), GPER1 siRNA (#SR301912, OriGene), cSrc siRNA (#SR304574, OriGene, Rockville, MD, USA) and p65 siRNA (#6534, Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Transfection, Plasmid Preparation

Journal: Journal of Bio-X Research

Article Title: GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

doi: 10.1097/jbr.0000000000000010

Figure Lengend Snippet: Figure 5. c-Src/NF-kB-p65 cascade is involved in the phosphorylation of FAK induced by E2. (A) SK-BR-3 cells were exposed to 10nM E2 for 20minutes, in presence or absenceofthePI3KinhibitorLY294002(LY;50mM),theMEKinhibitorPD98059(PD;5mM),thec-SrcinhibitorPP2(10mM),theGproteininhibitorPTX(100ng/mL)ortheNF-kB inhibitor PDTC (10mM). Top, Western blot of FAK and p-FAK; bottom, p-FAK densitometry values were adjusted to FAK intensity and then normalized to expression from the control sample. ∗P<.05, ∗∗P<.01, versus control; ##P<.01, versus E2. (B, C) SK-BR-3 breast cancer cells were treated with E2 (B) or G1 (C) at the indicated concentrations. Top, Western blot of c-Src, Tyr416-phosphorylated c-Src (p-c-Src), p65 or Ser536-phophor-p65; bottom, densitometry values of p-c-Src and p-p65 were adjusted to c-Src and p65 intensities, respectively, andthen normalized to expression from the controlsample. ∗P<.05, ∗∗P<.01, versus control. (D) SK-BR-3 cells were treated with 10nM E2 for 20 minuteswithorwithoutc-SrcinhibitorPP2(10mM)orNF-kBinhibitor PDTC (10mM).Top:Westernblotanalysis;bottom,p-c-Srcandp-p65densitometryvalues wereadjusted to c-Src and p65 intensities, respectively, then normalized to expression from the control sample. ∗P<.05, ∗∗P<.01, versus control; ##P<.01, versus E2. (E, F) SK-BR-3 cells were transfected with 100nM scrambled siRNA or target siRNAs for c-Src (E) or p65 (F) for 48hours, followed by treatment with E2 (10nM) or G1 (1mM) for 20minutes. Top, WesternblotforphosphorylationofFAK,c-Srcandp65;bottom,densitometricanalysisofphosphorylatedFAK,c-Srcandp65normalizedbyb-actin. ∗P<.05, ∗∗P<.01,versus control; ##P<.01,versuscorrespondingE2orG1inscrambledsiRNAgroup.Alltheexperimentswererepeatedatleast3timeswithconsistentresultsandrepresentativeimages were shown. NF-kB=nuclear factor kappa B, FAK: focal adhesion kinase, E2=17b-estradiol, GPER1=G-protein-coupled estrogen receptor.

Article Snippet: The siRNAs used for experiments included ERa siRNA (#sc29305, Santa Cruz, CA), ERb siRNA (#SR301462, OriGene, Rockville, MD, USA), GPER1 siRNA (#SR301912, OriGene), cSrc siRNA (#SR304574, OriGene, Rockville, MD, USA) and p65 siRNA (#6534, Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Transfection

Journal: Journal of Bio-X Research

Article Title: GPER1 promotes estrogen receptor negative breast cancer cell migration and invasion via non-genomic activation of c-Src/NF-κB/focal adhesion kinase cascade

doi: 10.1097/jbr.0000000000000010

Figure Lengend Snippet: Figure 6. GPER1 interacts with c-Src to promote cell migration and invasion. SK-BR-3 cells were transfected with 100nM GPER1 siRNAs or scrambled siRNAs for 48hours, followed by treatment with E2 (10nM) or GPER1 agonist G1 (1mM) for 20minutes. Left, Western blot; right, densitometry of phosphorylated FAK, c-Src and p65. ∗P<.05, ∗∗P<.01, versus control; #P<.05, ##P<.01, versus corresponding E2 or G1 in scrambled siRNA group. (B) SK-BR-3 cells were treated with vehicle, E2 or G1 for 20minutes and lysates were subjected to immunoprecipitation using anti-GPER1 antibody followed by Western blot with anti-c-Src antibodies. (C) Cells were transfected with 100nM GPER1 siRNAs or scrambled siRNAs for 48hours and then treated with E2 (10nM) or G1 (1mM) for an additional 48hours. Cell migration distances are shown below images as mean migration distance (mm)±SD. ∗∗∗P<.001, versus control; ###P<.001, versus E2 or G1 in scrambled siRNA group. (D) Cell invasion was assayed using invasion chambers. Representative images and invasion indexes are shown. ∗∗∗P<.001, vs. control; ###P<.001, versus E2 or G1 in scrambled siRNA group. All the experiments were repeated at least 3 times with consistent results. GPER1=G-protein-coupled estrogen receptor; FAK=focal adhesion kinase.

Article Snippet: The siRNAs used for experiments included ERa siRNA (#sc29305, Santa Cruz, CA), ERb siRNA (#SR301462, OriGene, Rockville, MD, USA), GPER1 siRNA (#SR301912, OriGene), cSrc siRNA (#SR304574, OriGene, Rockville, MD, USA) and p65 siRNA (#6534, Cell Signaling Technology).

Techniques: Migration, Transfection, Western Blot, Control, Immunoprecipitation

Journal: Oncogene

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

doi: 10.1038/onc.2015.391

Figure Lengend Snippet: Figure 3. mSH3BGRL activates MAPK and AKT pathways in an FAK-dependent manner. (a) Lysates from stable cell lines were incubated with GST-Raf to enrich for GTP-bound active Ras. Eluates and total lysates were subsequently immunoblotted with various Ras/MAPK pathway-specific antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (b) Lysates from (a) were immunoblotted with anti-p-FAK Y576/577 or FAK antibodies. (c) Lysates prepared as in (a) were incubated with GST-PBD (to enrich for GTP-bound active Cdc42 and Rac1). Elutes and total lysates were subsequently immunoblotted with antibodies against Cdc42 and Rac1. (d) CT-26 Vec, CT-26 SH3BGRL, CHO Vec and CHO SH3BGRL cells were transfected with FAK-specific siRNA, where indicated, for 48 h and lysed. Lysates were immunoblotted with the various antibodies listed. GAPDH was served as a loading control. The averaged relative protein expression level is quantified and shown under the immediate panels.

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, Incubation, Control, Transfection, Expressing

Journal: Oncogene

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

doi: 10.1038/onc.2015.391

Figure Lengend Snippet: Figure 4. 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 × 106 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, ***Po0.001).

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: Knockdown, Stable Transfection, Expressing, shRNA, Control, Clone Assay, Injection

Journal: Cancer Research

Article Title: The ShcA PTB Domain Functions as a Biological Sensor of Phosphotyrosine Signaling during Breast Cancer Progression

doi: 10.1158/0008-5472.can-12-4178

Figure Lengend Snippet: Figure 5. The ShcA R175Q-PTB mutant promotes VEGF production in an Src-dependent manner. A, VEGF ELISA on conditioned media from NT/ShcA or NT/ShcR175Q cells transfected with scrambled or Src-specific siRNAs (n ¼ 8 wells each). B, VEGF ELISA on conditioned media from NT/ShcA and NT/ShcR175Q cells were treated with DMSO alone or 20 mmol/L PP2 for 12 or 24 hours (n ¼ 6 wells each). C and D, whole- cell extracts were generated from the cells described in A and B and were subjected to immunoblot analysis with the indicated antibodies. All statistical analysis was conducted using a 2-tailed Student t test.

Article Snippet: The plugs were excised 8 days later and embedded in OCT. All animal studies were approved by the Animal Resources Centre (ARC) at McGill University and comply with guidelines set by the Canadian Council of Animal Care. siRNA/inhibitor studies For the siRNA studies, cells were transfected with a mixture of 3 dicer-substrate–duplex siRNAs targeting mouse Src (Origene-SR416929) or with a universal scrambled control (Origene-SR30004) using INTERFERin transfection reagents (Cat# 409-10; Polyplus). siRNAs were used at a final concentration of 60 nmol/L per transfection (20 nmol/L for each individual ShcA siRNA).

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Transfection, Generated, Western Blot

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