Figure 3. Biochemical effects of EGFR and PI3K pathway combined inhibition.

• A
  MDA‐MB‐468 cells were treated for 24 h with vehicle, PI3Kβi (AZD8186 250 nM), EGFRi (gefitinib 3 μM), or cetuximab (100 μg/ml), alone or in combination. The cell lysates were probed with the indicated antibodies.
• B
  MDA‐MB‐468 cells were treated for 24 h with vehicle, AKTi (MK2206 450 nM), gefitinib (3 μM), or cetuximab (100 μg/ml), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.
• C, D
  HCC70 (C) and ZR‐75‐1 (D) parental cells or PI3Kβi‐Res (derivative cells with acquired resistance to AZD8186), or AKTi‐Res (acquired resistance models to MK2206) were treated with vehicle, PI3Kβi (AZD8186 250 nM), or AKTi (MK2206 1 μM) for 24 h. The whole cell lysates were then probed with the indicated antibodies. Spliced images of parental and resistant paired samples were taken from the same original blot, and blots showing P‐S6 and S6 tot in (D) have been cut and reassembled for figure purposes.
• E
  HCC70 parental, PI3Kβi‐Res, or AKTi‐Res cells were treated with vehicle, AKTi (MK2206 250 nM), or gefitinib (3 μM), alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.
• F
  HCC70 MK res (acquired resistance models to MK2206) were treated with serial dilutions of gefitinib, alone, or in combination with MK2206 (810 nM), as indicated, and viability measured after 4 days of treatment. Average ± SD of triplicates and representative of two independent experiments.
• G
  HCC70 AZD res (acquired resistance models to AZD8186) were treated with serial dilutions of gefitinib, alone, or in combination with AZD8186 (270 nM), as indicated, and viability measured after 4 days. Average ± SD of triplicates and representative of two independent experiments.
• H, I
  p110β co‐immunoprecipitates with EGFR in MDA‐MB‐468 (H) and in HCC70 (I). Cells were pre‐treated with different inhibitors, including vehicle, pan‐PI3Ki GDC0941 (1 μM for MDA‐MB‐468 or 0.5 μM for HCC70), EGFRi (gefitinib 3 μM), PI3Kβi (AZD8186 250 nM), or a combination of EGFRi and PI3Kβi. Cell lysates were incubated with IgG control or anti‐EGFR antibody, and the immuno‐complexes or the total lysates were immune‐blotted with the indicated antibodies.
• J
  EGF‐induced increase in phospho‐AKT is dependent on p110β kinase activity. MDA‐MB‐468 were starved in 0% FBS and pre‐treated with vehicle or PI3Kβi (AZD8186 250 nM) for 1 h. Cell lysates were probed with the indicated antibodies. Phospho‐AKT and pan‐AKT bands were quantified by the use of ImageLite software: The ratio of phospho‐AKT to pan‐AKT normalized to the control (left hand lane) is shown.

Source data are available online for this figure.