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. Author manuscript; available in PMC: 2016 Jun 15.
Published in final edited form as: Cancer Res. 2015 Apr 13;75(12):2489–2500. doi: 10.1158/0008-5472.CAN-14-3167

Figure 3. Determining the functional role of KRAS gain in acquired resistance to EGFR inhibitors.

Figure 3

(A) Immunoblotting of PC9 AR_1 (KRAS gain) cells grown in 1.5μM afatinib transfected with 20nM of NRAS, KRAS or control siRNA for 48 hours. (B) PC9 AR_1 (KRAS gain) cells grown in 1.5μM afatinib treated for 96 hours with 20nM of NRAS, KRAS or control siRNA. Cell number was determined by nuclei count. (C) PC9 GR_1 (EGFR T790M / KRAS gain) cells grown in 1.5μM gefitinib were transfected with 20nM of KRAS or control siRNA −/+ 160nM AZD9291. After 4 days cell number was determined by nuclei count. Data shown is representative data. Error bars are standard deviation. (D) Immunoblotting of PC9 GR_1 cells, grown in media containing gefitinib, transfected with 20nM of KRAS or NTC siRNA for 5 days and then treated with 160nM of AZD9291 for 2 hours. (E) Immunoblotting of PC9 AR_1 (KRAS gain) cells grown in 1.5μM afatinib and (F) WZR_3 (KRAS gain) cells grown in 1.5μM WZ4002 transfected with 20nM of KRAS or control siRNA for 48 hours and then treated for 4 hours +/− 500nM selumetinib. (G) PC9 AR_1 (KRAS gain) cells grown in 1.5μM afatinib and (H) WZR_3 (KRAS gain) cells grown in 1.5μM WZ4002 treated for 96 hours with 20nM of KRAS or control siRNA +/− 500 nM selumetinib. Cell number was determined by nuclei count.