* indicates p 0.05 by student’s t-test. (E) Long-term viability assays with the indicated agents BYL719 1 M (BYL), GDC-0941 1 M (GDC), LEE011 1 M (LEE), or the indicated combination were performed in mutant cell lines with resistance to PI3K inhibition. Glo as described by the manufacturer. For all experiments data shown are mean of triplicates, with error bars representing -/+ SEM. Figure S2 (related to Figure 2): Viability curves of sensitizers from combinatorial drug screen. (A) Resistant cell lines were treated with escalating doses of each of 42 targeted agents, in the absence or presence of a fixed dose of PI3K inhibitor. Proliferation was assessed after 5 days using Cell Titer Glo. Viability values for each curve were normalized to the measured inhibition value at zero dose of the respective targeted agent without or with 1 M PI3Ki. The agents were scored based on difference in AUC between the two curves (minus and plus PI3K inhibitor) and ranked by greatest difference. Agents with FDR values 0.05 were considered sensitizers in the drug screen for individual cell lines. The viability curves for the sensitizers in each of the resistant cell lines are depicted as means of triplicates -/+ SEM. (B) Structure of CDK 4/6 inhibitor, LEE011. (C) Biochemical Assay of LEE011 to determine relative IC50 against CDK 4 and other CDKs. Figure S3 (related to Figure 3): Combination of PI3K inhibitors with MK2206 or lapatinib (A) Proliferation was assessed by Cell Titer Glo in parental and resistant 453 and T47D lines treated with escalating doses of MK2206 for 5 days. Resistant lines were also treated with escalating doses of MK2206 in the presence of 1 M GDC-0941, and dose response curves were generated. Values were normalized to the measured inhibition value at zero dose of MK2206 (without or with GDC-0941 1 M for resistant lines). (B) Parental and resistant 453 and T47D cells were treated for 24 hours with escalating doses of MK2206. 453R and T47DR cells were also treated with increasing doses of MK2206 in the presence of GDC0941 1 M. Lysates were prepared at 24 hours and probed with the indicated antibodies. (C) Long-term viability assays were performed on parental and resistant 453 lines with BYL719 1 M, lapatinib 1 M, or the combination. Media was changed every 72 hours. When the untreated controls grew to confluence, the cells in both the vehicle and drug treated wells were fixed and stained for nucleic acid with Syto-60. To assess viability, quantification of absorbance in the Syto-60 stained cells was performed using infrared imaging. *indicates p 0.05 by student’s t test. (D) Parental and resistant 453 cells were treated for 24 hours with escalating doses of lapatinib. 453R cells were also treated with increasing doses of lapatinib in the presence of BYL719 1 M. Lysates were prepared at 24 hours and probed with the indicated antibodies. Error bars in this figure represent mean +/-SEM of triplicates. Figure S4 (related to Figure 4): Synergy scores between PI3K and CDK4/6 inhibition in sensitive and resistant mutant cell lines. (A) A 67 dose matrix of either GDC-0941 (GDC) or BYL719 (BYL) and LEE011 (LEE) was performed and cell viability was measured Mouse Monoclonal to Rabbit IgG (kappa L chain) at the end of 5 days using Cell Titer Glo. Loewe Excess Inhibition values were calculated as a measure of synergy and are displayed. Values greater than 10% were considered to be suggestive of synergy. Calculation of Loewe Excess Inhibition is described in Supplemental Experimental Procedures. (B) PI3Ki resistant cells were treated with escalating doses of PD-0332991 (PD) in the absence or presence of a fixed 1 M dose of PI3Ki. Proliferation was assessed after 5 days using Cell Titer Glo. Viability values for each curve were normalized to the measured inhibition value at zero dose of PD (without or with PI3Ki for the resistant lines). (C) Long-term viability assays were performed on parental and resistant lines with BYL719 or GDC-0941 1 M (BYL or GDC as indicated), PD0332991 1 M (PD), or the indicated combination. Media was changed every 72 hours. When the untreated controls grew to confluence, the cells in both the vehicle and drug treated wells were fixed and stained for nucleic acid with Syto-60. To assess viability, quantification of absorbance in the Syto-60 stained cells was performed using infrared imaging. *indicates p 0.05 by student’s t test. (D) Transient knockdown using scrambled control siRNA (or and was performed in MCF7R,.*indicates Febuxostat (TEI-6720) p 0.05 by student’s t test. (C-D) Cells were treated with escalating doses of either BYL719 or GDC-0941, as indicated (C) or BEZ235 (D) as indicated for 72 hours. Viability was assessed using Cell Titer Glo as described by the manufacturer. (E) Cells were treated with escalating doses of the indicated agents for 120 hours. Viability was assessed using Cell Titer Glo as explained by the manufacturer. For all experiments data demonstrated are mean of triplicates, with error bars representing -/+ SEM. Number S2 (related to Number 2): Viability curves of sensitizers from combinatorial drug display. (A) Resistant cell lines were treated with escalating doses of each of 42 targeted providers, in the absence or presence of a fixed dose of PI3K inhibitor. Proliferation was assessed after 5 days using Cell Titer Glo. Viability ideals for each curve were normalized to the measured inhibition value at zero dose of the respective targeted agent without or with 1 M PI3Ki. The providers were scored based on difference in AUC between the two curves (minus and plus PI3K inhibitor) and rated by very best difference. Providers with FDR ideals 0.05 were considered sensitizers in the drug display for individual cell lines. The viability curves for the sensitizers in each of the resistant cell lines are depicted as means of triplicates -/+ SEM. (B) Structure of CDK 4/6 inhibitor, LEE011. (C) Biochemical Assay of LEE011 to determine relative IC50 against CDK 4 and additional CDKs. Number S3 (related to Number 3): Combination of PI3K inhibitors with MK2206 or lapatinib (A) Proliferation was assessed by Cell Titer Glo in parental and resistant 453 and T47D lines treated with escalating doses of MK2206 for Febuxostat (TEI-6720) 5 days. Resistant lines were also treated with escalating doses of MK2206 in the presence of 1 M GDC-0941, and dose response curves were generated. Values were normalized to the measured inhibition value at zero dose of MK2206 (without or with GDC-0941 1 M for resistant lines). (B) Parental and resistant 453 and T47D cells were treated for 24 hours with escalating doses of MK2206. 453R and T47DR cells were also treated with increasing doses of MK2206 in the presence of GDC0941 1 M. Lysates were prepared at 24 hours and probed with the indicated antibodies. (C) Long-term viability assays were performed on parental and resistant 453 lines with BYL719 1 M, lapatinib 1 M, or the combination. Media was changed every 72 hours. When the untreated settings grew to confluence, the cells in both the vehicle and drug treated wells were fixed and stained for nucleic acid with Syto-60. To assess viability, quantification of absorbance in the Syto-60 stained cells was performed using infrared imaging. *shows p 0.05 by student’s t test. (D) Parental and resistant 453 cells were treated for 24 hours with escalating doses of lapatinib. 453R cells were also treated with increasing doses of lapatinib in the presence of BYL719 1 M. Lysates were prepared at 24 hours and probed with the indicated antibodies. Error bars with this number symbolize mean +/-SEM of triplicates. Number S4 (related to Number 4): Synergy scores between PI3K and CDK4/6 inhibition in sensitive and resistant mutant Febuxostat (TEI-6720) cell lines. (A) A 67 dose matrix of either GDC-0941 (GDC) or BYL719 (BYL) and LEE011 (LEE) was performed and cell viability Febuxostat (TEI-6720) was measured at the end of 5 days using Cell Titer Glo. Loewe Febuxostat (TEI-6720) Extra Inhibition values were calculated like a measure of synergy and are displayed. Values greater than 10% were considered to be suggestive of synergy. Calculation of Loewe Extra Inhibition is explained in Supplemental Experimental Methods. (B) PI3Ki resistant cells were treated with escalating doses of PD-0332991 (PD) in the absence or presence of a fixed 1 M dose of PI3Ki. Proliferation was assessed after 5 days using Cell Titer Glo. Viability ideals for each curve were normalized to the measured inhibition value at zero dose of PD (without or with PI3Ki for the resistant lines). (C) Long-term viability assays were performed on parental and resistant lines with BYL719 or GDC-0941 1 M (BYL or GDC as indicated), PD0332991 1 M (PD), or the indicated combination. Media was changed every 72 hours. When the untreated settings grew to confluence, the cells in both the vehicle and.