Journal: Oncogene

Article Title: Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

doi: 10.1038/onc.2012.97

Figure Lengend Snippet: Figure 1. IGF-I upregulates GPER expression in MCF-7 and Ishikawa cells. (a, b) IGF-I induces GPER mRNA expression, as evaluated by real- time PCR. The mRNA expression of GPER was normalized to 18S expression. Results are shown as fold changes of mRNA expression upon treatment compared to cells treated with vehicle (). (c, d) GPER protein levels were evaluated by immunoblotting in cells treated for 24 h with 100 ng/ml IGF-I. (e, f) GPER protein expression was evaluated by immunoblotting in cells treated for 24 h with vehicle () or 100 ng/ml IGF-I alone and in combination with 10 mM IGF-IR inhibitor tyrphostin AG1024 (AG), 10 mM PKC inhibitor GF109203X (GF), 10 mM PKCd inhibitor Rottlerin (Rot), 10 mM MEK inhibitor PD98059 (PD), 10 mM PKA inhibitor H89, 10 mM PI3K inhibitor LY294,002 (LY), as indicated. (g--j) The upregulation of GPER protein levels by 100 ng/ml IGF-I was abrogated in the presence of shIGF-IR. Side panels show densitometric analysis of the blots normalized to b-actin. Each column represents the mean±s.d. of three independent experiments performed in triplicate. J, K, ’ Indicate Po0.05 for cells receiving vehicle () versus treatments.

Article Snippet: Membranes were blocked and probed with primary antibodies against GPER (N-15), CTGF (L-20), c-Fos (H-125), phosphorylated ERK 12 (E-4), and ERK2 (C-14), phosphorylated PKCd (Thr 507), PKCd (C-20), cyclin D1 (M-20), ERa (F-10), IGF-IR (7G11), and b-actin (C2) purchased from Santa Cruz Biotechnology (DBA), and p-ERaSer118(16J4) purchased from Cell Signaling Technology.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncogene

Article Title: Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

doi: 10.1038/onc.2012.97

Figure Lengend Snippet: Figure 3. Transduction pathways mediating GPER upregulation by IGF-I in MCF-7 cells. Immunoblots of p-PKCd (a) and p-ERK1/2 (b) from MCF-7 treated for 15 min with vehicle () or 100 ng/ml IGF-I alone and in combination with 10 mM PKC inhibitor GF109203X (GF), 10 mM PKCd inhibitor Rottlerin (Rot), 10 mM MEK inhibitor PD98059 (PD). Immunoblots shown are representative of experiments performed in triplicate. Side panels show densitometric analysis of the blots normalized to total ERK2 and PKCd. (c) Immunoblotting of c-fos from MCF-7 cells treated for 3 h with vehicle () or 100 ng/ml IGF-I alone and in combination with 10 mM IGF-IR inhibitor tyrphostin AG1024 (AG), 10 mM PKC inhibitor GF109203X (GF), 10 mM PKCd inhibitor Rottlerin (Rot), 10 mM MEK inhibitor PD98059 (PD). (d) Cells were transfected with AP1-luc-responsive collagenase promoter and treated with 100 ng/ml IGF-I alone and in combination with AG, GF, Rot or PD, as indicated. (e) The expression vector encoding for a dominant negative form of c-fos (DN/c-fos) blocked the transactivation of AP1-luc by 100 ng/ml IGF-I. (f) A 100-ng/ml IGF-I induces the recruitment of c-fos to the AP1 site located within the GPER promoter sequence. In control samples, non-specific IgG was used instead of the primary antibody. (g) The expression vector encoding for a dominant negative form of c-fos (DN/c-fos) blocked the transactivation of the GPER promoter construct. (h) The expression vector encoding for a dominant negative form of c-fos (DN/c-fos) blocked the upregulation of GPER protein levels by 100 ng/ml IGF-I. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle () were set as onefold induction upon which the activities induced by 100 ng/ml IGF-I were calculated. Each column represents the mean±s.d. of three independent experiments performed in triplicate. K, ’, &, J Indicate Po0.05 for cells receiving vehicle () versus treatments. Side panels in (c) and (h) show densitometric analysis of the blots normalized to b-actin.

Article Snippet: Membranes were blocked and probed with primary antibodies against GPER (N-15), CTGF (L-20), c-Fos (H-125), phosphorylated ERK 12 (E-4), and ERK2 (C-14), phosphorylated PKCd (Thr 507), PKCd (C-20), cyclin D1 (M-20), ERa (F-10), IGF-IR (7G11), and b-actin (C2) purchased from Santa Cruz Biotechnology (DBA), and p-ERaSer118(16J4) purchased from Cell Signaling Technology.

Techniques: Transduction, Western Blot, Transfection, Expressing, Plasmid Preparation, Dominant Negative Mutation, Sequencing, Control, Construct, Luciferase

Journal: Oncogene

Article Title: Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

doi: 10.1038/onc.2012.97

Figure Lengend Snippet: Figure 5. GPER, CTGF and ERa are involved in the migration of (a) MCF-7 and (b) Ishikawa cells induced by IGF-I. Cell migration promoted by IGF-I was abolished silencing GPER, CTGF or ERa expression. Bar graph shows a representative experiment with means of triplicate samples, standardized to the respective untreated controls set to100%. Error bars show standard deviations. J Indicates Po0.05 for cells receiving vehicle () versus treatments.

Article Snippet: Membranes were blocked and probed with primary antibodies against GPER (N-15), CTGF (L-20), c-Fos (H-125), phosphorylated ERK 12 (E-4), and ERK2 (C-14), phosphorylated PKCd (Thr 507), PKCd (C-20), cyclin D1 (M-20), ERa (F-10), IGF-IR (7G11), and b-actin (C2) purchased from Santa Cruz Biotechnology (DBA), and p-ERaSer118(16J4) purchased from Cell Signaling Technology.

Techniques: Migration, Expressing

Journal: Oncogene

Article Title: Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

doi: 10.1038/onc.2012.97

Figure Lengend Snippet: Figure 6. GPER is required for proliferation of MCF-7 and Ishikawa cells induced by IGF-I. (a, c) Cell proliferation induced by 100 ng/ml IGF-I was abrogated by silencing GPER expression. (e, g) The upregulation of cyclin D1 protein by 100 ng/ml IGF-I was abolished in the presence of shGPER and DN/c-fos. Side panels show densitometric analysis of the blots normalized to b-actin. (b, d, f) Efficacy of GPER silencing. (h, i) The treatment for 24 h with 100 ng/ml IGF-I strongly increases the coimmunoprecipitation of GPER with cyclin D1 in MCF-7 cells, as indicated. In control samples, non-specific IgG was used instead of the primary antibody. Each column represents the mean±s.d. of three independent experiments performed in triplicate. J, K, ’, & Indicate Po0.05 for cells receiving vehicle () versus treatments.

Article Snippet: Membranes were blocked and probed with primary antibodies against GPER (N-15), CTGF (L-20), c-Fos (H-125), phosphorylated ERK 12 (E-4), and ERK2 (C-14), phosphorylated PKCd (Thr 507), PKCd (C-20), cyclin D1 (M-20), ERa (F-10), IGF-IR (7G11), and b-actin (C2) purchased from Santa Cruz Biotechnology (DBA), and p-ERaSer118(16J4) purchased from Cell Signaling Technology.

Techniques: Expressing, Control

Journal: Oncogene

Article Title: Insulin-like growth factor-I regulates GPER expression and function in cancer cells.

doi: 10.1038/onc.2012.97

Figure Lengend Snippet: Figure 7. ERa is involved in the GPER transcription by IGF-I. (a) The transactivation of GPER promoter construct induced by 100 ng/ml of IGF-I is abrogated in presence of 10 mM ICI 182,780 (ICI). The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle () were set as onefold induction upon which the activity induced by treatments was calculated. (b, c) The IGF-I induced upregulation of GPER protein levels was abolished by 10 mM ICI and by silencing ERa expression. (d) Efficacy of ERa and p-ERaSer118 silencing. (e, f) The recruitment of p-ERaSer118 induced by 100 ng/ml IGF-I to the AP1 site located within the GPER promoter sequence is abolished in presence of an expression vector encoding a dominant negative form of c-fos (DN/c-fos). In control samples non-specific IgG was used instead of the primary antibody. Each column represents the mean±s.d. of three independent experiments. &, K, J Indicate Po0.05 for cells receiving vehicle () versus treatments.

Article Snippet: Membranes were blocked and probed with primary antibodies against GPER (N-15), CTGF (L-20), c-Fos (H-125), phosphorylated ERK 12 (E-4), and ERK2 (C-14), phosphorylated PKCd (Thr 507), PKCd (C-20), cyclin D1 (M-20), ERa (F-10), IGF-IR (7G11), and b-actin (C2) purchased from Santa Cruz Biotechnology (DBA), and p-ERaSer118(16J4) purchased from Cell Signaling Technology.

Techniques: Construct, Luciferase, Transfection, Control, Activity Assay, Expressing, Sequencing, Plasmid Preparation, Dominant Negative Mutation

Journal: Poultry science

Article Title: Effects of maternal hawthorn-leaf flavonoid supplementation on the intestinal development of offspring chicks.

doi: 10.1016/j.psj.2024.103969

Figure Lengend Snippet: Figure 2. Effects of maternal dietary HF on the protein expression. mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3 kinase; Akt, protein kinase B; PCNA, proliferating cell nuclear antigen; IGF-1R, insulin-like growth factor 1 receptor. CON, mother generation fed with a basic corn-soybean diet; LHF, mother generation fed with a basic corn-soybean diet supplemented with 30 mg/kg hawthorn-leaf flavonoid; HHF, mother generation fed with a basic corn-soybean diet supplemented with 60 mg/kg hawthorn-leaf flavonoid. Data are presented as mean value § SEM (n = 6). Bars with different letters (a, b) indicate significant difference (P < 0.05).

Article Snippet: The primary antibodies were PCNA (1:1000, ab29, Abcam, Cambridge, UK), p-mTOR (1:1000, AP0490, ABclonal Biotechnology Co., Ltd., Wuhan, China), mTOR (1:1000, A2445, ABclonal Biotechnology Co., Ltd., Wuhan, China), p-AKT (1:1000, 966l, Cell Signaling), AKT (1:1000, 13038s, Cell Signaling, USA), PI3K (1:1000, AB2757501, ABclonal Biotechnology Co., Ltd., Wuhan, China), IGF-1R (1:1000, 20254-1- AP, Proteintech Group, Inc., IL, USA), b-actin (1:5000, 66009, Proteintech Group, Inc., IL).

Techniques: Expressing