NRF1 is fundamental for ubiquitin-proteasome pathway function. (A) STRING⁷⁰ enrichment regulatory network identified among the 8 NRF1 dependent genes associated with the ubiquitin pathway. Biological process and KEGG pathway analysis of 8 NRF1 dependent genes associated with the ubiquitin pathway (right). (B) Heat map depicting RNA-seq Kms18 cells at siControl (left) and siNRF1 (right). Color scheme: red for higher z score, and blue for lower z score of TPM. (C) Relative mRNA levels (qRT-PCR) of the indicated genes in Kms18 MM cell line transiently transfected with siNRF1 or siControl (left). Values were normalized to actin expression. Error bars represent the standard error of 3 separate experiments. Student t test ∗P < .05; ∗∗∗P < .005; ∗∗∗∗P < .001. Representative WB analysis of Kms18 MM cell line TCEs transfected as above and probed for the indicated antibodies (right). (D) Total cell lysates from Kms18 cell depleted or not for NRF1 expression were subjected to immunoblot analysis with indicated antibodies and Ponceau staining. (E) The analysis of relative mRNA levels (qRT-PCR) (left) and WB analysis (right) for the indicated genes in Kms18 MM cell line transiently transfected with siNRF1 or siControl. Values were normalized to actin expression. Error bars represent the standard error of 3 separate experiments. Student t test ∗P < .05; ∗∗∗∗P < .001. (F) qRT-PCR (left) and WB analysis (right) of Kms18 MM cells stably expressing the dCas9-KRAB transcriptional repressor complex and transiently transfected with control (scramble) or targeting sgRNAs (NRF1) for the expression levels of indicated genes and antibodies. Values were normalized to actin expression. Error bars represent the standard error of 3 separate experiments. Student t test ∗P < .05; ∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001. (G) Immunoblot analysis of TCE from dCas9-KRAB Kms18 MM cells transiently transfected as in panel F and analyzed for the indicated antibodies and Ponceau staining. (H) qRT-PCR (left) and WB analysis (right) of Kms18 MM cells stably expressing the dCas9-KRAB transcriptional repressor complex and transiently transfected as in panel F for the expression levels of indicated genes and antibodies. Values were normalized to actin expression. Error bars represent the standard error of 3 separate experiments. Student t test ∗P < .05; ∗∗P < .01; ∗∗∗P < .005. (I) Proliferation assay (left), qRT-PCR (middle), and WB analysis (right) of Kms18 MM cells transiently transfected with siControl or si-NRF1-3′UTR and MycNRF1 as indicated. Data are presented as the mean ± SD of 3 independent experiments and error bars represent the SD. Values were normalized to actin expression. Analysis of variance (ANOVA) test (∗∗P < .01; ∗∗∗P < .005; ∗∗∗∗P < .001). (J) Kms18 MM cells transfected as above and analyzed for Ponceau staining, semi-qPCR (u/s XBP1), WB, and qRT-PCR for antibodies and genes specified. Data are presented as the mean ± SD of 3 independent experiments, with values normalized to actin expression. Error bars represent the SD. ANOVA test ∗∗P < .01; ∗∗∗∗P < .001. (K) Proteasome activity in Kms18 and Kms27 MM cells transiently transfected with siControl or siNRF1. Proteasome-specific chymotryptic, trypsin-like, and caspase-like activities were assessed in cell extracts and expressed on a per-protein basis. The histogram shows the relative quantification of all 3 activities within each line. The average of at least 3 independent experiments (SD) is shown. Student t test, not significant. (L) Protein degradation of Kms18 MM cells transiently transfected with siControl or siNRF1. The cells were pulsed for 30 minutes with ³⁵S amino acids and chased for the indicated times with or without MG132 (4 μM). The data showed indicate the percentage of trichloroacetic acid–insoluble radioactivity, the disappearance of which was inhibited by MG132 at any given time point, relative to the total radioactivity present at the end of the pulse. Error bars represent the standard error of 2 separate experiments. Student t test ∗P < .05. BP, biological process; KEGG, Kyoto Encyclopedia of Genes and Genomes; mRNA, messenger RNA; N°, number of cells; sgControl, single-guide control; u/s XBP1, unspliced/spliced XBP1.