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. 2022 Oct 23;82(21):3962–3973. doi: 10.1158/0008-5472.CAN-21-4339

Figure 2.

Figure 2. SMG8 and SMG9 deficiency causes resistance to ATR inhibition. A, YCC6 SMG8 mutant clones show lower levels of SMG8 protein expression compared with SMG8 WT YCC6 cells by Western blotting. B, YCC6 SMG9 mutant clones show no SMG9 protein expression compared with SMG9 WT YCC6 cells by immunoprecipitation. C–F, SMG8 and SMG9 mutant clones are resistant to ATRi (384-well plate, 5-day assay), compared with the WT cells. G, Western blot analysis showing doxycycline-inducible overexpression of HA-tagged SMG8 cDNA in SMG8 Mut 1 clone. Doxycycline-inducible GFP cDNA overexpression serves as a negative control. H and I, ATRi dose–response survival curves (384-well plate, 5-day assay) illustrating a resensitization to berzosertib and AZD6738 in the SMG8 Mut 1 clone when SMG8 overexpression was induced by doxycycline exposure (dotted line, compared with the continuous line) J, SMG8 (blue) and SMG9 (red) mutant HAP1 cells are resistant to ATRi (384-well plate, 5-day assay). All panels of this figure are representative of three or more biological replicates.

SMG8 and SMG9 deficiency causes resistance to ATR inhibition. A, YCC6 SMG8 mutant clones show lower levels of SMG8 protein expression compared with SMG8 WT YCC6 cells by Western blotting. B, YCC6 SMG9 mutant clones show no SMG9 protein expression compared with SMG9 WT YCC6 cells by immunoprecipitation. C–F, SMG8 and SMG9 mutant clones are resistant to ATRi (384-well plate, 5-day assay) compared with the WT cells. G, Western blot analysis showing doxycycline-inducible overexpression of HA-tagged SMG8 cDNA in SMG8 Mut 1 clone. Doxycycline-inducible GFP cDNA overexpression served as a negative control. H and I, ATRi dose–response survival curves (384-well plate, 5-day assay) illustrating a resensitization to berzosertib and AZD6738 in the SMG8 Mut 1 clone when SMG8 overexpression was induced by doxycycline exposure (dotted line, compared with the continuous line). J, SMG8 (blue) and SMG9 (red) mutant HAP1 cells are resistant to ATRi (384-well plate, 5-day assay). All panels of this figure are representative of three or more biological replicates.