Figure 5. Inhibition of Erk1/2 phosphorylation by X-370 contributed to its antiproliferative activity.

(A-C) X-370 inhibited Erk1/2 phosphorylation via PI3K-PDK1-MEK1/2 cascade but not the PI3K-Gab1 positive feedback loop. (A) Raji and SU-DHL-6 cells were exposed to 1 μM X370 for 3 h and protein fraction from cellular membrane was separated using Native Membrane Protein Extraction Kit and probed. WCL: whole cell lysate. (B) Inhibition of PDK1 or MEK1/2 abrogated Erk1/2 phosphorylation. Cells were treated with pharmacological inhibitors of Raf (AZ 628, PLX4032), MEK1/2 (AZD6244), p90RSK (BI-D1870), PI3Kδ (X-370), PDK1 (BX-912), Akt (MK-2206), mTOR (AZD8055) or mTORC1 (Rapamycin) respectively and indicated proteins were detected. (C) X-370 disrupted the association between PDK1 and MEK1/2. Raji cells were treated with 1 μM X370 for 1 h and cell lysates were immunoprecipitated with anti-PDK1 antibody and indicated proteins were detected. WCL: whole cell lysate. (D-F) Inhibition of Erk1/2 phosphorylation was required for the anti-proliferative activity of X-370. (D) Raji cells ectopically expressing a constitutively activated phospho-mimic MEK1 mutant (MEK1 S202D/S204D or MEK DD) were incubated with X-370 or AZD6244 for 1 h and then subjected to Western blot. (E) Raji and Raji MEK DD cells were treated with X-370 for 72 h and cell proliferation was detected by CCK-8 assay. (F) Raji MEK DD cells were treated with X-370 and AZD6244 and cell proliferation was detected by CCK-8 assay. Data were show as mean ± SD of three independent experiments. *: p < 0.05 determined by t-tests at each data point.