1. Date of download: 7/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Imatinib-Mediated Inactivation of Akt Regulates ABCG2 Function in Head and Neck Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2008;134(9):979-984. doi:10.1001/archotol.134.9.979 Downregulation of Akt decreases Hoechst 33342 extrusion. Hoechst staining analysis of cells treated with Akt kinase inhibitors LY294002 (B), imatinib mesylate (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) (C), and 1L6- hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (H) was performed. The effects of short interfering RNA Akt (E) and transfection of a dominant negative Akt plasmid (F) were also analyzed. Density dot plot analyses (Hoechst red vs Hoechst blue) are shown with the percentage of cells gated for high ABCG2 activity. The first results of each set of data are the control samples, to which the treatments were compared correspondingly (A, D, and G). The side population was decreased with the addition of verapamil hydrochloride. Figure Legend:

2. Date of download: 7/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Imatinib-Mediated Inactivation of Akt Regulates ABCG2 Function in Head and Neck Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2008;134(9):979-984. doi:10.1001/archotol.134.9.979 Reduction of doxorubicin efflux with Akt inhibition. We incubated UMSCC10B cells in doxorubicin hydrochloride for 1 hour at 37°C, followed by 2.5 hours of extrusion. Cells were treated with imatinib mesylate (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) and 1L6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (1L6), and were transfected with short interfering RNA (siRNA) before fluorescence-activated cell sorter analysis. Intracellular levels of doxorubicin were measured with or without extrusion, demonstrating the validity of the assay (A). The table (B) displays the percentage of change of intracellular doxorubicin levels for the 3 different treatments compared with untreated cells. Figure Legend:

3. Date of download: 7/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Imatinib-Mediated Inactivation of Akt Regulates ABCG2 Function in Head and Neck Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2008;134(9):979-984. doi:10.1001/archotol.134.9.979 Akt regulates localization of ABCG2 but not protein levels. Western blot analysis demonstrates a decrease in phospho-Akt levels when UMSCC10B cells are treated with LY294002 and imatinib mesylate (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) and transfected with short interfering RNA (siRNA) Akt (A). An Akt kinase activity assay performed on UMSCC10B cells treated with 0μM, 5μM, and 10μM imatinib shows a dose-dependent decrease in Akt kinase activity (B). Total levels of ABCG2 were not significantly altered when cells were treated with imatinib or LY294002, but immunoprecipitated cell surface ABCG2 levels decreased with downregulation of Akt (C). Immunofluorescence showed translocation of ABCG2 (green) from the cell surface toward the nucleus when treated with imatinib, LY294002, and 1L6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O- octadecylcarbonate (1L6) (D). Figure Legend:

4. Date of download: 7/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Imatinib-Mediated Inactivation of Akt Regulates ABCG2 Function in Head and Neck Squamous Cell Carcinoma Arch Otolaryngol Head Neck Surg. 2008;134(9):979-984. doi:10.1001/archotol.134.9.979 Treatment with imatinib mesylate (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, New Jersey) sensitizes HNSCC cells to doxorubicin hydrochloride in a clonogenic survival assay. UMSCC10B (A) and HN30 (B) cells were treated with 2μM or 6μM imatinib, respectively, and exposed to varying doses of doxorubicin for 7 to 10 days. Cell survival was estimated from the number of colonies with more than 50 cells. The results showed that imatinib with doxorubicin decreased colony survival more than doxorubicin alone. Experiments were performed at least in duplicate. Figure Legend: