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. 2019 Nov 19;8:e50223. doi: 10.7554/eLife.50223

Figure 6. Mechanistic insights into ARIH2 loss-mediated EGFR-TKI resistance.

(A) Mass spectrometry analysis of global protein changes between control and ARIH2-deficient HCC827 cells. (B) Immunoblots of indicated proteins in control and ARIH2-deficient HCC827 cells. β-Actin was used as a loading control. (C) Quantitative RT-PCR analysis of relative mRNA levels of indicated genes in control or ARIH2-deficient HCC827 cells. Error bars represent mean ± SD; n = 4. (D) Immunoblots of indicated proteins showing ectopic expression of HA-METAP2 in HCC827 cells. GAPDH was used as a loading control. (E) Crystal violet staining colony formation assay of HCC827-Vector or HCC827-HA-METAP2 cell lines treated with DMSO, erlotinib (1 µM), or gefitinib (1 µM). (F) Quantification of colony formation in (E), shown as percentage of the HCC827-Vector sample. Mean (three biological replicates) ± SD is shown. (G) De novo protein synthesis of HCC827-Vector or HCC827-HA-METAP2 cells after treatment with DMSO or erlotinib (1 µM, 24 hr) as determined by L-azidohomoalanine (AHA) labeling. Cells were starved of methionine for 1 hr and incubated with AHA for 1 hr. Lysates were subjected to a Click-iT chemistry reaction to switch azido-modified nascent proteins to alkyne-biotin, and visualized by Streptavidin-HRP immunoblotting. β-Actin was used as a loading control. (H) De novo protein synthesis of control or ARIH2-deficient HCC827 cells after treatment with DMSO or erlotinib (1 µM, 24 hr) as determined by AHA labeling. β-Actin was used as a loading control. (I) Immunoblot of METAP2 in HCC827 cells upon proteasome inhibitor bortezomib treatment. β-Actin was used as a loading control. (J) Immunoblots of ARIH2 and METAP2 in control or ARIH2 knockout HCC827 cells upon bortezomib treatment. β-Actin was used as a loading control. (K) De novo METAP2 protein synthesis in control or ARIH2-deficient HCC827 cells as determined by AHA labeling and streptavidin pulldown. β-Actin was used as a loading control. (L) Immunoblots of indicated proteins showing ectopic expression of HA-ALDOA and HA-PSAT1 in HCC827 cells. GAPDH was used as a loading control. (M) Crystal violet staining colony formation assay of HCC827-Vector, HCC827-HA-ALDOA or HCC827-HA-PSAT1 cells treated with DMSO, erlotinib (1 µM), or gefitinib (1 µM). (N) Quantification of colony formation in (M), shown as percentage of the HCC827-Vector sample. Mean (three biological replicates) ± SD is shown. Statistical significance was tested using unpaired two-tailed t test (C, F and N); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant.

Figure 6—source data 1. Raw data from Figure 6.

Figure 6.

Figure 6—figure supplement 1. ARIH2 loss increases protein abundance of METAP2, ALDOA and PSAT1.

Figure 6—figure supplement 1.

(A) Immunoblots of indicated proteins in control and ARIH2-deficient NCI-H3255 cells. β-Actin was used as a loading control. (B) Quantitative RT-PCR analysis of relative mRNA levels of indicated genes in control or ARIH2-deficient NCI-H3255 cells. Error bars represent mean ± SD; n = 4. (C) Immunoblots of indicated proteins in control and ARIH2- or CUL5-deficient HEK293T cells. HSP90 was used as a loading control. (D) Immunoblots of indicated proteins in a panel of EGFR-mutant and EGFR-WT NSCLC cell lines and the normal human bronchial epithelial cell line BEAS-2B. β-Actin was used as a loading control. (E) Immunoblots of indicated proteins in HCC827 cells upon erlotinib treatment. β-Actin was used as a loading control. (F) Immunoblots of indicated proteins in control or ARIH2 knockout HCC827 cells upon erlotinib treatment. β-Actin was used as a loading control. (G) Detection of endogenous METAP2 protein ubiquitination in HCC827 cells using the TUBE assay. Control or ARIH2 knockout HCC827 cells were treated with 0.1 µM bortezomib for overnight and harvested for TUBE assay.