Fig. 2

NOP56 depletion evokes IRE1α-mediated UPR. A, NOP56 depletion led to significant enrichment of the UPR gene signature in KRAS-mutant cancer cells. GSEA was based on the GEO dataset GSE15212. B, Transcriptional quantification (qRT-PCR) of UPR genes in H358 and H460 cells expressing control (sh Scram) or NOP56-specific shRNA (sh NOP56a). C, Immunoblots of H358 and H460 cells expressing scrambled control or NOP56-specific shRNAs. D, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with IRE1α-specific or control siRNAs for 72 h before immunoblotting. E, Clonogenic assay of H460 and H358 cells expressing scrambled control or NOP56-specific shRNAs after treated with indicated doses of 4μ8C (IRE1α inhibitor). Representative images are shown. F, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with IRE1α-specific or control siRNAs for 72 h, in the presence or absence of NAC (2.5 mM) before apoptosis assay. Data are presented as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two-way ANOVA with Tukey’s multiple comparisons test. G, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with IRE1α-specific or control siRNAs for 72 h, in the presence or absence of NAC (2.5 mM). Cells were then washed, incubated with H2DCFDA for 30 min, and analyzed by flow cytometry. Quantification of relative ROS levels was shown in the right. Data are presented as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two-way ANOVA with Tukey’s multiple comparisons test. H, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with HSF1-specific or control siRNAs for 72 h before apoptotic assay. I, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with HSF1-specific or control siRNAs for 72 h before immunoblot analysis. Data are presented as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two-way ANOVA with Tukey’s multiple comparisons test. J, Clonogenic assay of H460 and H358 cells expressing scrambled control or NOP56-specific shRNAs after treated with the indicated doses of KRIBB11 (HSF1 inhibitor). Representative images are shown. K, L, Clonogenic assay of H460 and H358 cells expressing scrambled control or NOP56-specific shRNAs after treated with the indicated doses of the ER stress inducer bortezomib (K) or tunicamycin (L). Representative images are shown