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: 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

Journal: Food Science and Human Wellness

Article Title: Sea cucumber derived sulfated sterols alter glucose metabolism by promoting gluconeogenesis and reducing glycogenesis in healthy mice

doi: 10.26599/fshw.2024.9250046

Figure Lengend Snippet: Fig. 4 Effects of sea cucumber sulfated sterols on protein expression level of hepatic insulin signals in mice (n = 6). (A) The protein expression of InsR, p-InsR, AKT and p-AKT were measured by Western blotting. (B) InsR/actin. (C) p-InsR/InsR. (D) AKT/actin. (E) p-AKT/AKT. Different letters indicate significant difference at P < 0.05 among the other three groups except for group N determined by ANOVA (Tukey’s test) and * indicate significant difference at P < 0.05 compared to the N group determined by Student’s t-test.

Article Snippet: All primary antibodies against insulin receptor (InsR) (#bs-0681R), p-InsR (#bs-16680R), protein kinase B (AKT) (#9272), p-AKT (#4060), glycogen synthase kinase-3 beta (GSK3β) (#bs-0023M), p-GSK3β (#bs-5367R), forkhead box protein O1 (FOXO1) (#9454), p-FOXO1 (#9461), GLUT4 (#bs-0384R), Na, K-ATPase (#3010), β-actin (#4967) and secondary antibodies were obtained from Cell Signaling Technology (Beverly, USA) and BIOSS (Beijing, China).

Techniques: Expressing, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Cyclocarya paliurus (Batal.) Ijinskaja Aqueous Extract (CPAE) Ameliorates Obesity by Improving Insulin Signaling in the Hypothalamus of a Metabolic Syndrome Rat Model

doi: 10.1155/2017/4602153

Figure Lengend Snippet: CPAE activates the insulin-signaling pathway in the hypothalamus of SHR/cp rats . (a) The phosphorylation level of InsR, (b) the phosphorylation level of IRS1tyr989, (c) the level of m-PI3Kp85, (d) the phosphorylation level of Akt, (e) the phosphorylation level of FoXO1, (f) the protein expression level of POMC, and (g) NPY of SHR/cp rats treated with 0.5 g/kg CPAE or untreated controls. All of the results were determined by Western blot analysis as described in Materials and Methods. Values are expressed as the mean ± SD. ∗ P < 0.05 and ∗∗ P < 0.01 versus control group. n = 6 per group.

Article Snippet: After being blocked using either Blocking One or Blocking One-p buffer (Nacalai Tesque, Kyoto, Japan), the membranes were incubated at 4°C overnight with the primary antibodies, including p-InsR (cat#, 3024s), PI3Kp85 (4257s), p-Akt (4060s), p-FoXO1(9461s), and β -actin antibodies (4970s) (Cell Signaling Technology, USA), as well as POMC (ab94446), NPY (ab180809) (Abcam, Eugene, OR, USA), and p-IRS1tyr989 (L1912) (Santa Cruz Biotechnology Inc., Dallas, TX, USA).

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