Extended Data Fig. 10: Molecular and functional studies of POU2AF1 and ER-associated dependencies.
a-b, Immunoblotting analyses to confirm that protein levels of POU2AF1 are decreased with Doxy-inducible CRISPR interference (a, KMS11 cells) and increased with CRISPR activation (b, LP-1 cells) compared to cells with sgRNAs for control OR genes. Beta-actin a, or vinculin b, were probed as loading controls in the same respective membrane concurrently with POU2AF1. c, Relative numbers of viable LP-1 cells with CRISPR-based activation of POU2AF1 vs. a control OR gene (day 12 after end of transduction with sgRNAs for POU2AF1; results qualitatively concordant with those at later time-point in Fig. 6b). CTG assay, mean +/− s.e.m. results; n = 6 independent replicate cell cultures per condition; one-way ANOVA and Tukey’s post-hoc test (detailed results included in Source Data), p < 0.001 for each POU2AF1 sgRNA vs. OR12D2 sgRNA). d, Immunoblotting for UBE2J1 after doxy-inducible CRISPR-based KO of UBE2J1 (or a control OR gene). Vinculin was probed as loading control concurrently with the staining for UBE2J1. Each experiment in a-d was performed once. e, UBE2J1, its dislocon complex partners SEL1L, SYVN1, and other ER-related MM preferential dependencies are among the top ‘hits’ in two genome-scale screens (using retroviral gene-trap mutagenesis and CRISPR gene-editing)45 for genes involved in ERAD regulation (in KBM7 haploid cells). f, In vitro bortezomib treatment (24 h) of KMS18 cells with Doxy-inducible CRISPR KO of SYVN1 or control OR genes. (CTG; mean +/− s.e.m.; n = 8 independent replicate cell cultures for drug-free control and n = 4 independent replicate cell cultures per drug dose for each KO; 2-way ANOVA (p < 0.001); detailed results of Tukey post-hoc tests included in Source Data). g-h, Patterns of CERES scores in MM (n = 19) and non-MM (n = 770) lines for g, ER/ERAD/Golgi-related genes and h, select ER genes. Results are presented similar to format of Fig. 1. Highlighted gene symbols include MM-preferential dependencies (red); examples of core essential genes (green); and genes which do not meet all criteria for MM-preferential dependencies but are recurrently essential for MM cell lines and are linked with the function of the ER glycoprotein quality control system (blue) and the ER translocon system (purple).
