Journal:

Article Title: Insulin-like growth factor I receptor blockade enhances chemotherapy and radiation responses and inhibits tumour growth in human gastric cancer xenografts

doi: 10.1136/gut.2004.048926

Figure Lengend Snippet: Expression and roles of the insulin-like growth factor/insulin-like growth factor I receptor (IGF/IGF-Ir) axis in human gastric cancer cell lines. (A) Representative data of reverse transcription-polymerase chain reaction analyses. Both MKN45 and MKN74 cells expressed IGF-I message (396 bp) but neither MKN28 nor NUGC4 showed any expression. All cells expressed mRNAs of IGF-II (468 bp), IGF-Ir (755 bp), and insulin-like growth factor receptor 2 (IGF-2r) (430 bp). Controls were β-actin (540 bp) and GAPDH (300 bp). (B–E) After MKN45 cells were cultured to 60% confluence (1×106) in six well plates, medium was replaced with the indicated medium (complete medium (CM) or serum free medium (SFM)) for an additional 48 hours and then cell growth evaluated by trypan blue assay. Growth ratio was calculated compared with the cell number in complete medium with 10% fetal calf serum (1.76 (0.05)×106). Cell growth was suppressed by serum withdrawal (SFM, p<0.0001) but 100 ng/ml IGF-I ((B); p = 0.0230, SFM without IGF-I v SFM with IGF-I) and 100 ng/ml IGF-II ((C); p = 0.0227, SFM without IGF-II v SFM with IGF-II) partially restored growth. (D) Insulin-like growth factor binding protein 3 (IGFBP3) reduced cell growth in complete medium. (E) The growth suppressing effect of IGFBP3 was also seen in serum free medium dose dependently. (F–H) Both IGF-I (F) and IGF-II (100 ng/ml (G, H)) blocked induction of 5% ethanol (EtOH one hour) induced apoptosis in both MKN45 (F, G) and MKN74 (H). (F) p<0.0001, no stimulation v ethanol stimulation without IGF-I; p = 0.0003, ethanol without IGF-I v ethanol with 200 ng/ml IGF-I. (G) p = 0.0097, no stimulation v ethanol stimulation without IGF-II; p = 0.0179, ethanol without IGF-I v ethanol with IGF-I. (H) p = 0.0022, no stimulation v ethanol stimulation without IGF-II; p = 0.0033, ethanol without IGF-I v ethanol with IGF-I.

Article Snippet: Recombinant human IGF-I and IGF-II were purchased from R&D systems (Minneapolis, Minnesota, USA) and NH2 terminally truncated IGF-I (des(1–3) IGF-I) from GroPep (Adelaide, Australia).

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Cell Culture, Binding Assay

Journal:

Article Title: Insulin-like growth factor I receptor blockade enhances chemotherapy and radiation responses and inhibits tumour growth in human gastric cancer xenografts

doi: 10.1136/gut.2004.048926

Figure Lengend Snippet: Downstream signals from insulin-like growth factor I receptor (IGF-Ir) in gastric cancer cells, MKN45 (A–F), NUGC4 (G, H), and MKN74 (I, J). (A) In MKN45, western blotting showed that 20 ng/ml insulin-like growth factor I (IGF-I) phosphorylated Akt-1, extracellular signal regulated kinase (ERK), and Bad. Both Akt and Bad phosphorylation was reduced by dominant negative forms (dns) of adenoviruses expressing IGF-Ir (Ad-IGF-Ir/dns, 30 multiplicity of infection (moi)). However, Ad-IGF-Ir/dns did not influence ERK-1/-2 phosphorylation to the same degree as Akt. pAkt, phosphorylated Akt-1; tAkt, total Akt-1; pERK, phosphorylated ERK-1/-2; tERK, total ERK-1/-2; pBad, phosphorylated Bad; tBad, total Bad. (B) Western blotting revealed that 10 ng/ml IGF-II phosphorylated Akt which was reduced by infection with adenoviruses expressing truncated IGF-Ir of 482 amino acids long (Ad-IGF-Ir/482st) (30 moi). (C) IGF-Ir/482st blocked p38 activity by p38 kinase assay. ATF2 is a substrate of p38 MAPK. (D) Insulin induces Akt phosphorylation which was not influenced by IGF-Ir/482st. (E) NH2 terminally truncated IGF-I (des(1-3)IGF-I 20 ng/ml) phosphorylated Akt to the same degree as IGF-I. Ad-IGF-Ir/482st reduced des(1-3)IGF-I inducing Akt phosphorylation. dIGF-I, des(1-3)IGF-I. (F) IGF-Ir/482st did not block Akt phosphorylation stimulated by high dose IGF-I, especially 200 ng/ml IGF-I. (G) In NUGC4, IGF-I stimulated phosphorylation of Akt and p38 mitogen activated protein kinase (MAPK) were blocked by both IGF-Ir/dns (30 moi) but phosphorylation of ERKs were not. p-p38, phosphorylated p38 MAPK; t-p38, total p38 MAPK. (H) IGF-Ir/dn blocked IGF-II induced Akt phosphorylation. (I, J) In MKN74, IGF induced Akt phosphorylation was blocked by 100 moi of Ad-IGF-Ir/482st, but phosphorylated ERK was not. LacZ, control (adenovirus expressing β-galactosidase).

Article Snippet: Recombinant human IGF-I and IGF-II were purchased from R&D systems (Minneapolis, Minnesota, USA) and NH2 terminally truncated IGF-I (des(1–3) IGF-I) from GroPep (Adelaide, Australia).

Techniques: Western Blot, Phospho-proteomics, Dominant Negative Mutation, Expressing, Infection, Activity Assay, Kinase Assay, Blocking Assay, Control

Journal: Cell Discovery

Article Title: VAPB-mediated ER-targeting stabilizes IRS-1 signalosomes to regulate insulin/IGF signaling

doi: 10.1038/s41421-023-00576-6

Figure Lengend Snippet: a The gel lane was subjected to trypsin digestion followed by MALDI-TOF analysis, and VAPB was identified as a potential interacting partner of IRS-1. Peptides identified by MS analysis of FLAG-IRS-1 immunoprecipitates were listed. b C2C12 myoblasts, treated with or without 100 ng/mL IGF-1 for 2.5 min after serum starvation for 16 h, were subjected to immunoprecipitation with IRS-1 antibodies. Coimmunoprecipitated IRS-1 and VAPB were detected by Western blot analysis. The relative IRS-1 levels co-precipitated by VAPB were quantified. * P < 0.05. c FLAG-tagged IRS-1 or 9YA mutant was co-transfected with HA-VAPB into 293 T cells for co-IP analysis. d GST or GST-VAPB fusion protein was incubated with purified FLAG-tagged IRS-1 for direct Pull-down assay. e Schematic diagram of VAPB and its mutants. f FLAG-tagged IRS-1 or mutant constructs as shown in ( e ) were co-transfected with GFP-IRS-1 into 293 T cells for co-IP assays. g FLAG-tagged IRS-1 was co-transfected with HA-VAPB or VAPB KMDD mutant into 293 T cells for co-IP analysis.

Article Snippet: C2C12 cells were serum-starved for 12 h in DMEM, and then treated with 100 ng/mL IGF-1 (Sino biological, 10598-HNAY1).

Techniques: Immunoprecipitation, Western Blot, Mutagenesis, Transfection, Co-Immunoprecipitation Assay, Incubation, Purification, Pull Down Assay, Construct

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet: In vitro differential suppression of podocyte IGF1R activity reveals that partial inhibition is beneficial but near total loss is highly detrimental (A) Representative Western blot shows >90% reduction of IGF1R protein in NC-IGF1RKD cells but no reduction of IR protein expression. Bar graphs show densitometry expressed as the mean fold change +/− SEM, t-test, ∗∗∗∗ p < 0.0001, n = 18 independent experiments. (B) Phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min was significantly reduced in NC-IGF1RKD podocytes. Data are expressed as the mean ± SEM, one-way ANOVA with Tukey’s multiple comparison test, ∗∗ p < 0.005, n = 3 independent experiments. (C) Western blot shows that IGF1R expression is reduced by ∼70% in wild-type podocytes exposed to 100 nM picropodophyllin for 24 h. Data are expressed as the mean ± SEM, t-test, ∗ p < 0.05, n = 3 independent experiments. No significant change in IR expression was observed. (D) Western blot shows the phosphorylation of AKT and p44/42MAPK in response to acute IGF1 stimulation at 10 ng and 100 ng/mL for 10 min in podocytes exposed to 100 nM picropodophyllin for 24 h. Data expressed as the mean ± SEM, ∗ p < 0.05, n = 3 independent experiments. (E) ∼50% of NC-IGF1RKD cells survive 7 days after gene excision. Treatment of wild-type podocytes with 100 nM picropodophyllin for 24 h has no effect on cell survival. Data are expressed as the mean ± SEM, t-test, ∗∗ p < 0.005, n = 3–4 independent experiments. See also Figures S5 and .

Article Snippet: Recombinant Mouse IGF1 protein , Novus , Cat# NBP2-35081.

Techniques: In Vitro, Activity Assay, Inhibition, Western Blot, Expressing, Comparison

Journal: iScience

Article Title: Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition

doi: 10.1016/j.isci.2024.109749

Figure Lengend Snippet:

Article Snippet: Recombinant Mouse IGF1 protein , Novus , Cat# NBP2-35081.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Quantitation Assay, Staining, RNAscope, Western Blot, Cell Culture, Software, Plasmid Preparation