(A) Immunoblot of ARIH2 confirms the ARIH2 knockout efficiency in HCC827 cells. HSP90 was used as a loading control. (B) Cell viability assessment by CellTiter-Glo assay of control or ARIH2 knockout HCC827 cells treated with serial dilutions of erlotinib for 72 hr. Error bars represent mean ± SD; n = 4. (C) Activated caspase 3/7 measurement of control or ARIH2 knockout HCC827 cells treated with serial dilutions of erlotinib for 24 hr. Error bars represent mean ± SD; n = 4. (D) Kinetic cell proliferation assay monitored by IncuCyte for indicated HC827 cell lines cultured in the presence of DMSO control or 1 µM erlotinib over a 9 day period. (E) Drug-tolerant persister (DTP) cells were generated by treating control and ARIH2 knockout HCC827 cells with 1 µM of erlotnib or gefitinib for 9 d. Percentage of DTP cells is shown relative to DMSO-treated cells. Error bars represent mean ± SD; n = 3. (F) Crystal violet staining colony formation assay of control or ARIH2 knockout HCC827 cell lines treated with DMSO, erlotinib (1 µM), or gefitinib (1 µM). (G) Quantification of colony formation in (F), shown as percentage of the sgAAVS sample. Error bars represent mean ± SD; n = 3. (H) Schematic outline of the competitive proliferation assay to assess the selective outgrowth of ARIH2 knockout HCC827 cells upon EGFR-TKI treatment. RFP-negative HCC827 cells were spiked with approximately 1% RFP-positive sgRNA-infected HCC827 cells, control (sgAAVS) or ARIH2 knockout (sgARIH2), and grown for 3 weeks in the absence or presence of 1 µM erlotinib or gefitinib. Cells were collected and analyzed for RFP positivity by FACS. (I) Selective outgrowth of ARIH2 knockout cells in the presence of EGFR-TKI in HCC827 cell line. The percentage of RFP-positive cells is indicated. FSC, forward scatter. (J) Quantification of the selective outgrowth of ARIH2 knockout cells in the presence of EGFR-TKI as shown in (I). Mean (three biological replicates) ± SD is shown. (K) ARIH2 knockout promotes acquired resistance to erlotinib in HCC827 xenograft model. Mice bearing HCC827 xenografts, control (sgAAVS) or ARIH2 knockout (sgARIH2), were dosed once daily with 10 mg/kg erlotinib or vehicle for the indicated time frame. Data are represented as mean tumor volume (mm3) ± s.e.m., n = 8 mice for each line. (L) Percentage change in tumor volume compared to baseline (the start of dosing, day 13 post-implantation) for individual cell xenografts treated for 29 d (day 42 post-implantation) with vehicle or 64 d (day 77 post-implantation) with 10 mg/kg erlotinib. Statistical significance was tested using unpaired two-tailed t test (E, G and L) or ordinary two-way ANOVA (J); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.