Journal: NPJ Systems Biology and Applications

Article Title: Mapping the dynamics of insulin-responsive pathways in the blood–brain barrier endothelium using time-series transcriptomics data

doi: 10.1038/s41540-022-00235-8

Figure Lengend Snippet: a , b Scatter plot of cells’ calcein/EthD-1 fluorescence showing the percentage of total counted cells within the live and dead gates in a untreated control and b insulin-treated (300 minutes) cells. c Immunoblots showing IR-β expression in hCMEC/D3 cells treated with insulin for 40 and 300 minutes. d Dynamic changes of insulin receptor gene (INSR) expression in hCMEC/D3 cells treated with insulin up to 300 minutes.

Article Snippet: The proteins were then electroblotted onto a 0.45 μm nitrocellulose membrane, blocked with 5% nonfat dry milk protein (Bio-Rad Laboratories, Hercules, CA), and incubated overnight at 4 °C with primary antibodies against IR-β (1:1000, #3025, Cell Signaling Technology, Denvers, MA) and GAPDH (1:1000, #5174, Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Control, Western Blot, Expressing

Journal: Science signaling

Article Title: Wnt signaling regulates hepatic metabolism.

doi: 10.1126/scisignal.2001249

Figure Lengend Snippet: Fig. 3. β-Catenin modulates in vivo hepatic insulin signaling. (A) Assessment of IRS-1 and IRS-2 abundance and tyrosine phosphorylation in GFP control animals or Ad-Cre–infected mice (n = 3). Deletion of β-catenin results in higher basal tyrosine phosphorylation of IRS-1 and IRS-2. (B) Analysis of phosphorylation of Akt Ser473 and GSK-3β Ser9 under basal conditions or 5 min after insulin injection. Signaling was assessed in control GFP mice (n = 4 mice; 2 insulin-stimulated) or a similar number of Ad-Cre–infected animals. (C) Influence of β-catenin overexpression on in vivo insulin signaling. Insulin-stimulated tyrosine phosphorylation of the insulin receptor β chain (IRβ) and serine phosphorylation of GSK-3β were reduced in mice that overexpressed β-catenin. (D) In vivo assessment of de novo glycogen incorporation in control (GFP) or β-catenin–overexpressing mice. Both hepatic and skeletal muscle glycogen syntheses were assessed. n = 8 mice per group. All Western blots are representative of experiments that were performed at least three times. *P < 0.05, Student's t test. NS, not significant.

Article Snippet: For detection of insulin signaling intermediates, 1 mg of liver protein lysate was used for immunoprecipitation with antibodies directed against the insulin receptor (IR) β chain (Santa Cruz Biotechnology), IRS-1 (Santa Cruz Biotechnology), or IRS-2 (Santa Cruz Biotechnology), respectively.

Techniques: In Vivo, Phospho-proteomics, Control, Infection, Injection, Over Expression, Western Blot

Journal: BMC Cancer

Article Title: Insulin-like growth factor receptor and sphingosine kinase are prognostic and therapeutic targets in breast cancer

doi: 10.1186/s12885-017-3809-0

Figure Lengend Snippet: Immunohistochemistry and manual scoring analysis of Australian Breast Cancer Tissue Bank patient samples. Immunohistochemistry was performed on formalin-fixed paraffin embedded breast cancer patient tissue samples ( n = 236) obtained from the Australian Breast Cancer Tissue Bank (ABCTB) using antibodies to detect and measure relative levels of IGF1R, p-IGF1R and SphK1. The intensity of immunostaining was assessed by manual scoring according to standard guidelines as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining and 3 = strong staining for IGF1R, p-IGF1R and SphK1

Article Snippet: The sections were quenched in 0.3% hydrogen peroxide for 5 min, blocked with 5% goat serum for 30 min and incubated in primary antibodies: IGF1Rβ antibody (no cross-reaction with the insulin receptor (InsR)) (#3027, Cell Signaling, Danvers, MA, USA 1:100), p-IGF1R (#ab39398, Abcam, Melbourne, VIC, Australia, 1:200) and SphK1 (#AP7237c, Abgent, San Diego, CA, USA, 1:200) for one hour at room temperature.

Techniques: Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Immunostaining, Staining

Journal: BMC Cancer

Article Title: Insulin-like growth factor receptor and sphingosine kinase are prognostic and therapeutic targets in breast cancer

doi: 10.1186/s12885-017-3809-0

Figure Lengend Snippet: Survival outcomes in relation to p-IGF1R, IGF1R and/or SphK1 protein expression in breast cancer patients. Kaplan-Meier analysis was performed to measure the overall survival (OS) following stratification for high vs. low p-IGF1R, IGF1R and SphK1 protein expression as follows: a . IGF1R; b . p-IGF1R; c . IGF1R and SphK1 co-expression and d . p-IGF1R and SphK1 co-expression as described under Methods

Article Snippet: The sections were quenched in 0.3% hydrogen peroxide for 5 min, blocked with 5% goat serum for 30 min and incubated in primary antibodies: IGF1Rβ antibody (no cross-reaction with the insulin receptor (InsR)) (#3027, Cell Signaling, Danvers, MA, USA 1:100), p-IGF1R (#ab39398, Abcam, Melbourne, VIC, Australia, 1:200) and SphK1 (#AP7237c, Abgent, San Diego, CA, USA, 1:200) for one hour at room temperature.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Insulin-like growth factor receptor and sphingosine kinase are prognostic and therapeutic targets in breast cancer

doi: 10.1186/s12885-017-3809-0

Figure Lengend Snippet: Survival outcomes in relation to p-IGF1R, IGF1R and/or SphK1 protein expression in ER positive and negative breast cancer tissues. Kaplan-Meier analysis was performed to measure the overall survival (OS) following stratification for high vs. low IGF1R, SphK1 protein expression stratified for ER expression as follows: a . IGF1R (ER-positive); b . IGF1R and SphK1 co-expression (ER-positive); C. IGF1R (ER-negative) and D. IGF1R and SphK1 (ER-negative) as described under Methods

Article Snippet: The sections were quenched in 0.3% hydrogen peroxide for 5 min, blocked with 5% goat serum for 30 min and incubated in primary antibodies: IGF1Rβ antibody (no cross-reaction with the insulin receptor (InsR)) (#3027, Cell Signaling, Danvers, MA, USA 1:100), p-IGF1R (#ab39398, Abcam, Melbourne, VIC, Australia, 1:200) and SphK1 (#AP7237c, Abgent, San Diego, CA, USA, 1:200) for one hour at room temperature.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Insulin-like growth factor receptor and sphingosine kinase are prognostic and therapeutic targets in breast cancer

doi: 10.1186/s12885-017-3809-0

Figure Lengend Snippet: Survival outcomes in relation to IGF1R and/or SphK1 protein expression in hormone therapy treated breast cancer patients. Kaplan-Meier analysis was performed to measure the overall survival (OS) for ( a ). hormone therapy (anti-endocrine therapy) treatment (non-stratified) and further stratified for high vs. low ( b ). IGF1R ( c ). IGF1R and SphK1 co-expression and ( d ). SphK1 expression as described under Methods. Statistical significance was accepted as a log-rank p -value <0.05

Article Snippet: The sections were quenched in 0.3% hydrogen peroxide for 5 min, blocked with 5% goat serum for 30 min and incubated in primary antibodies: IGF1Rβ antibody (no cross-reaction with the insulin receptor (InsR)) (#3027, Cell Signaling, Danvers, MA, USA 1:100), p-IGF1R (#ab39398, Abcam, Melbourne, VIC, Australia, 1:200) and SphK1 (#AP7237c, Abgent, San Diego, CA, USA, 1:200) for one hour at room temperature.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Insulin-like growth factor receptor and sphingosine kinase are prognostic and therapeutic targets in breast cancer

doi: 10.1186/s12885-017-3809-0

Figure Lengend Snippet: Co-targeting IGF1R and SphK1 effects on cell viability and colony formation in breast cancer cells. a - d . MTT-assay and ( e - g ). colony formation experiments were performed using the ER-positive; MCF7 and T47D and ER-negative; HCC1806 and HCC70 breast cancer cell-lines. 2 × 10 3 cells were plated in either 96-well or 6-well plates, cultured for 24 h and subsequently treated with the dual IGF1R/InsR tyrosine kinase inhibitor (OSI-906; 0.1-6.4 μM) and/or SphK1inhibitor (SKI-II; 4 μM) for 96 h (MTT-assay) and 10-14 d (clonogenic assay). Repeated measures ANOVA was performed to determine the effect of SKI-II addition to OSI-906 does-response curves. Graphs depict experimental data normalized to zero treatment vehicle control and 1-way ANOVA followed by Tukey’s test was performed to determine significance between treatment groups and significance accepted p -values * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. All experiments were performed in triplicate for MTT-assay and duplicate for clonogenic assay. Note: The SKI 4 μM plus OSI 6.4 μM treatment was only performed in duplicate for the HCC1806 clonogenic assay experiments

Article Snippet: The sections were quenched in 0.3% hydrogen peroxide for 5 min, blocked with 5% goat serum for 30 min and incubated in primary antibodies: IGF1Rβ antibody (no cross-reaction with the insulin receptor (InsR)) (#3027, Cell Signaling, Danvers, MA, USA 1:100), p-IGF1R (#ab39398, Abcam, Melbourne, VIC, Australia, 1:200) and SphK1 (#AP7237c, Abgent, San Diego, CA, USA, 1:200) for one hour at room temperature.

Techniques: MTT Assay, Cell Culture, Clonogenic Assay, Control