Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Blautia Coccoides is a Newly Identified Bacterium Increased by Leucine Deprivation and has a Novel Function in Improving Metabolic Disorders.

doi: 10.1002/advs.202309255

Figure Lengend Snippet: Figure 5. I3AA has beneficial effects on metabolic improvements in vitro and in vivo. A) Primary hepatocytes were incubated with indicate dose of I3AA for 48 h and then stimulated with 100 nM insulin for 20 min (n = 6 replicates per group). Western blot analysis of p-IR, p-AKT, and p-GSK3𝛽levels. The right panel is the densitometry analysis of the relative abundance of phosphorylated proteins normalized to their total protein levels. A.U.: arbitrary units. B–J) HFD mice were orally gavage with PBS or 10 mg kg−1 I3AA for 4 weeks (n = 6–7 biological replicates per group). B) Fed and fasting blood glucose levels. C) Fed and fasting serum insulin levels assayed by ELISA. D) HOMA-IR index. E) Glucose tolerance tests. The right panel is the AUC. F) Insulin tolerance tests (0.5 U kg−1). The right panel is the AUC. G) Total fat mass. H) The H&E staining of sWAT. Scale bars, 50 μm. The bottom panel is the frequency distribution of adipocyte cell size in sWAT and the box plot is average adipocyte diameter. I) Real-time PCR analysis of browning related genes (Ucp1, Pgc1𝛼, Prdm16, and Cidea) in sWAT. J) Western blot analysis of UCP1 protein levels. The bottom panel is the densitometry analysis of UCP1 protein levels. All values are expressed as the mean ± SEM. Statistical comparisons were carried out by unpaired two-tailed Student’s t-test; *p < 0.05 and **p < 0.01.

Article Snippet: Protein samples were subjected to concentration determination and immunoblot assay with the following antibodies: phosphorylated (p)-IR (Tyr 1150/1151; 3024S), total IR (3025), p-AKT (Ser 473; 9271S), total AKT (9272S), p-GSK3β (Ser 9; 9336S), and total GSK3β(9315S) from Cell Signaling Technology; AhR (17840-1-AP) and β–Actin (81115-1-RR) from Proteintech; FGF21 (ab171941) and UCP1 (ab209483) from Abcam.

Techniques: In Vitro, In Vivo, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: IGF-IR A. , ERK B. and p-38 C. phosphorylation in cells transfected for 24 h with shRNA or shIGF-IR treated with vehicle (-) or 100 ng/ml IGF-I for 15 min. D.-E. Efficacy of IGF-IR silencing. ERK F. and p-38 G. activation in cells treated for 15 min with vehicle (-) or 100 ng/ml IGF-I alone and in combination with either 1 μM IGF-IR inhibitor tyrphostin AG1024 (AG), or 1 μM MEK inhibitor PD98059 (PD) or 1 μM p38 inhibitor SB202190 (SB). Side panels show densitometric analysis of the blots normalized to β-actin, ERK2 and p38 that served as loading controls respectively for pIGF-IR, pERK and p-p38. Data shown are the mean ± SD of three independent experiments. (◾) p < 0.05 for cells receiving vehicle (-) versus treatments.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Phospho-proteomics, Transfection, shRNA, Activation Assay

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: A. mRNA expression of GPER in cells treated with either vehicle (-) or 100 ng/ml IGF-I, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. B. GPER protein levels were evaluated by immunoblotting in cells treated with either vehicle (-) or 100 ng/ml IGF-I, as indicated. C. GPER protein expression in cells transfected for 24 h with either shRNA or shIGF-IR and then treated for 8 h with vehicle (-) or 100 ng/ml IGF-I. D.-E. Efficacy of IGF-IR silencing. F. Cells were transfected for 24 h with shRNA or shIGF-IR together with the GPER promoter construct. Then, cells were treated for 18 h with vehicle (-) or 100 ng/ml IGF-I. The luciferase activities were normalized to the internal transfection control, and values of cells receiving vehicle (-) were set as one fold induction upon which the activity induced by treatments was calculated. G. GPER protein levels in cells treated for 8 h with vehicle (-) or 100 ng/ml IGF-I alone or in combination with 1 μM IGF-IR inhibitor tyrphostin AG1024 (AG), 1 μM MEK inhibitor PD98059 (PD) and 1 μM p38 inhibitor SB202190 (SB). Side panels show densitometric analysis of the blots normalized to β-actin. H.-I. Co-immunoprecipitation studies performed in cells treated for 8 h with vehicle (-) or 100 ng/ml IGF-I, as indicated. In control samples, non-specific IgG was used instead of the primary antibody. H. Side panel show densitometric analysis of the blot normalized to IGF-IR. I. Side panel show densitometric analysis of the blot normalized to GPER. Data shown are the mean ± SD of three independent experiments. (◾) p < 0.05 for cells receiving vehicle (-) versus treatments.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Real-time Polymerase Chain Reaction, RNA Expression, Western Blot, Transfection, shRNA, Construct, Luciferase, Control, Activity Assay, Immunoprecipitation

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: (A-B) mRNA expression of CTGF and EGR1 in cells treated with either vehicle (-) or 100 ng/ml IGF-I, as evaluated by real-time PCR. Results obtained from experiments performed in triplicate were normalized for 18S expression and shown as fold change of RNA expression compared to cells treated with vehicle. CTGF C. and EGR1 D. protein levels were evaluated by immunoblotting in cells treated with vehicle (-) or 100 ng/ml IGF-I, as indicated. Side panels show densitometric analysis of the blots normalized to β-actin and each data point represents the mean ± SD of three independent experiments. (◾) p < 0.05 for cells receiving vehicle (-) versus treatments.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

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

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: A.-F. CTGF and EGR1 protein levels in cells transfected for 24 h with shRNA, shIGF-IR or shGPER and then treated for 8 h with either vehicle (-) or 100 ng/ml IGF-I. Efficacy of IGF-IR C.-D. and GPER G.-H. silencing. Side panels show densitometric analysis of the blots normalized to β-actin. I.-J. Cells were transfected for 24 h with shRNA, shIGF-IR or shGPER together with the CTGF or EGR1 promoter construct. Then, cells were treated for 18 h with vehicle (-) or 100 ng/ml IGF-I. K. Cells were transfected for 24 h with a dominant negative form of c-fos (DN/c-fos) together with the CTGF or EGR1 promoter construct. Then, cells were treated for 18 h with vehicle (-) or 100 ng/ml IGF-I. The luciferase activities were normalized to the internal transfection control, and values of cells receiving vehicle (-) were set as one fold induction upon which the activity induced by treatments was calculated. Data shown are the mean ± SD of three independent experiments. (◾) p < 0.05 for cells receiving vehicle (-) versus treatments.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Transfection, shRNA, Construct, Dominant Negative Mutation, Luciferase, Control, Activity Assay

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: NOTCH 1 protein levels in cells transfected for 24 h with shIGF-IR A. or shGPER D. and then treated for 8 h with vehicle (-) or 100 ng/ml IGF-I. Efficacy of IGF-IR B.-C. and GPER E.-F. silencing. Side panels show densitometric analysis of the blots normalized to β-actin. (◾) p < 0.05 for cells receiving vehicle (-) versus treatments.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Transfection

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: A. The migration of IST-MES 1 and A549 cells upon 8 h treatment with vehicle (-), 10 μg/ml COL1 or 100 ng/ml IGF-I alone and in combination with 1 μM DDR1 inhibitor (DDR1 IN), as evaluated by Boyden Chamber assay. B. The migration of IST-MES 1 and A549 cells induced by 8 h treatment with 100 ng/ml IGF-I was prevented knocking down IGF-IR and GPER expression, as evaluated by Boyden Chamber assay. Efficacy of IGF-IR C.-D. and GPER E.-F. silencing. Values represent the mean ± SD of three independent experiments. (⦁) indicates p < 0.05 for cells treated with vehicle (-) versus treatments.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Migration, Boyden Chamber Assay, Expressing

Journal: Oncotarget

Article Title: Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells

doi: 10.18632/oncotarget.10348

Figure Lengend Snippet: IGF-I stimulates the expression of GPER and its target genes, then IGF-IR and GPER trigger the IGF-I regulation of DDR1 target genes. The functional cross-talk of IGF-IR, GPER and DDR1 contributes to the chemotaxis and migration observed in cancer cells.

Article Snippet: Cells were processed according to a previously described protocol [ ] to obtain protein lysate that was electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against antiphosphotyrosine antibody (4G10) (Merck Millipore, Milan, Italy), IGF-IR (7G11), GPER (N-15), CTGF (L-20), phosphorylated ERK1/2 (E-4), ERK2 (C-14), NOTCH 1 (C-20), EGR1 (588), phosphorylated p-38 (D-8), p-38 (A-12), β-actin (C2), (Santa Cruz Biotechnology, DBA, Milan, Italy).

Techniques: Expressing, Functional Assay, Chemotaxis Assay, Migration

Journal: The FASEB Journal

Article Title: Cysteine‐rich 61 inhibition attenuates hepatic insulin resistance and improves lipid metabolism in high‐fat diet fed mice and HepG2 cells

doi: 10.1096/fj.202400860R

Figure Lengend Snippet: List of antibodies used for western blotting.

Article Snippet: Antibodies against Cyr61 (#39382), Sirt6 (#12486), phosphorylated (p)‐AMPKα (Thr172) (#2531), AMPKα (#2532), p‐insulin receptor (IR) β (Tyr1361) (#3023), IR β (#3025), insulin receptor substrate 1 (IRS‐1) (#2382), p‐ protein kinase B (Akt) (Ser473) (#9271), Akt (#9272), p‐ glycogen synthase kinase‐3 alpha/beta (GSK‐3α/β) (Ser21/9) (#8566), GSK‐3α/β (#5676), p‐ nuclear factor kappa B (NF‐κB) p65 (Ser536) (#3033), NF‐κB p65 (#4764), p‐stress‐activated protein kinase (SAPK)/ c‐Jun N‐terminal kinase (JNK) (Thr183/Tyr185) (#9251), SAPK/JNK (#9252), and α‐tubulin (#2144) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot