Figure S3.
Inhibition of macropinocytosis inhibits cell proliferation and increases cell death in ATM inhibitor-treated cells. Related to Fig. 3. (A) Ovcar8 cells were treated with the ATM inhibitor KU60019 (2 μM) or the macropinocytosis inhibitor EIPA (2 μM) alone and in combination for 3 d in RPMI-1640 + 5% FBS complete media, and dextran uptake was determined at the end of the experiment. n = 3/group, one of two experiments is shown. Data represent mean ± SD. *P < 0.05; one-way ANOVA. (B and C) Ovcar3 (B) or Ovcar10 (C) cells were treated with the ATM inhibitor KU60019 (2 μM) or the macropinocytosis inhibitor EIPA (2 μM) alone and in combination for 3 d in RPMI-1640 + 5% FBS complete media. Proliferation (left panel) was assessed by crystal violet staining. Apoptosis (right panel) was assessed by Annexin V/7AAD staining. n = 3/group, one of three experiments is shown. Data represent mean ± SD. *P < 0.01; one-way ANOVA. (D) Synergy analysis using Combenefit software using the LOEWE model. (E) Ovcar8 cells were treated with the ATM inhibitor KU60019 (2 μM) or the macropinocytosis inhibitor EIPA (1.25 μM) alone and in combination for 3 d in RPMI-1640 + 0.1% FBS complete media. n = 3/group, one of four experiments is shown. Data represent mean ± SD. *P < 0.005; one-way ANOVA. (F) Ovcar8 cells were treated with the ATM inhibitor KU60019 (2 μM) or the Rac1 inhibitor eHop-016 (1.75 μM) alone and in combination for 3 d in RPMI-1640 + 5% FBS complete media. Proliferation was assessed by crystal violet staining. One of four experiments is shown. Data represent mean ± SD. *P < 0.001; one-way ANOVA. (G) Ovcar8 cells were treated with the ATM inhibitor KU60019 (2 μM) or the Rac1 inhibitor eHop-016 (1.4 μM) alone and in combination for 2 d in RPMI-1640 + 0.1% FBS complete media. Proliferation was assessed by crystal violet staining. One of two experiments is shown. Data represent mean ± SD. *P < 0.0001; one-way ANOVA. (H) IMR90 fibroblasts (non-macropinocytotic) were treated with the macropinocytosis inhibitor EIPA (2 μM) for 3 d. Proliferation was assessed by crystal violet staining. Data represent mean ± SD. ns = not significant. Unpaired two-sided t test. One of two experiments is shown. (I) pChk2 and total Chk2 immunoblot analysis in all of the tumors. β-Actin was used as a loading control.