Fig 1: Treatment of CXCR1/2 inhibitor, repertaxin could reduce tamoxifen resistance. (a) The dosage-dependent effect of repertaxin on cell viability of MCF-10A. Non-cancerous breast epithelial cell line MCF-10A was used. The cells were treated with different concentrations of repertaxin for 96 h. MTT assay was performed. Results were shown as mean ± SD from 5 independent experiments. One-way ANOVA was employed. Bonferroni’s multiple comparison test was employed to determine the significance between 0 nM and other concentrations. Repertaxin could reduce tamoxifen resistance in (b) MCF-7-BQ, (c) ZR-75-BQ and (d) LCC2. The cells were co-treated with 4 µM of 4-OHT and 0.1 µM of repertaxin for 14 days. Clonogenic assay was performed. 0.01% crystal violet was used to stain the colonies. Results were shown as mean ± SD from 4 independent experiments. Student’s t-test was employed to determine statistical significance between DMSO and repertaxin treated groups. *** represents p < 0.001. (e) Repertaxin could suppress AKT and ERK1/2 activation on BQ overexpressing cells. MCF-7, MCF-7-BQ, ZR-75 and ZR-75-BQ cells were treated with 0.1 µM of repertaxin for 48 h. Western blot was used to determine the expression of the protein candidates. GAPDH was used as the loading control.
Fig 2: Schematic diagram of PI3K/AKT signalling cascade illustrating some downstream biomarkers under investigation. RTK: receptor tyrosine kinases. Arrows denote activation and bars for inhibition.
Fig 3: Reported PI3K and Akt inhibitors. Structures 1 and 2 are adapted from [17], 3 from [18], and 4 from [19].
Fig 4: Repertaxin could reverse tamoxifen resistance in vivo. (a) ZR-75-BQ cell line was employed to establish xenografts. The cells were implanted onto the mammary fat pad of nude mice. The mice received saline, tamoxifen (4-OHT; 500 mg; twice per week), repertaxin (15 mg/Kg; twice per week), tamoxifen + repertaxin (500 mg of 4-OHT + 7.5 mg/Kg of repertaxin; twice per week) and tamoxifen + repertaxin (500 mg of 4-OHT + 15 mg/Kg of repertaxin; twice per week). Repertaxin was delivered by subcutaneous injection. After 4 weeks of treatment, tumors were harvested. (b) The photo showed the effect of different treatments on tumor size. Results were shown as mean ± SD from 4 independent tumors. Two-way ANOVA was performed. Bonferroni’s multiple comparison test was employed to determine the significance between saline and other treatment groups at each time point. ** and *** represent p < 0.01 and p < 0.001, respectively. (c) Treatment of repertaxin could reduce the levels of activated AKT and ERK1/2 in the tumors. Proteins were harvested from the tumors. Western blot was performed to analyze the expression of the indicated protein candidates in 3 of the independent tumors. GAPDH was used as the loading control.
Fig 5: Hyperglycemia and hyperlipidemia blocks the Insulin-Inpp5f negative feedback loop.(A) Correlation between Inpp5f expression and Akt activity in HFD induced diabetic hearts. n = 12 (B) Overexpression of Inpp5f reduced Akt phosphorylation. H9C2 cells were infected with control and Inpp5f overexpressing adenovirus. Inpp5f, Akt, phosphorylated Akt and ß-actin expression were analyzed by Western blot. Representative data of triplicates. (C) Knockdown of Inpp5f increased Akt phosphorylation. H9C2 cells were transfected with scramble siRNA or siRNA against Inpp5f. Inpp5f, Akt, phosphorylated Akt and ß-actin expression were analyzed by Western blot. Representative data of triplicates. (D) H9C2 cells transfected with scramble siRNA or siRNA were conditioned with serum free, high glucose and FFA medium before Insulin treatment. Phosphorylated Akt and total Akt were detected by Western blot. (E) Knockdown of Inpp5f rescued the glucose/FFA mediated inhibition of 2-NBDG uptake. H9C2 cells transfected with scramble siRNA or siRNA were conditioned with serum free, high glucose and FFA medium before Insulin treatment. Fifteen minutes later, 2-NBDG uptake was monitored by flow cytometry.
Supplier Page from Abcam for Akt Kinase Activity Assay Kit