Figure 6.
NRF1 upregulation mediates adaptive resistance to proteasome inhibition. (A) qRT-PCR of the indicated genes (left) and WB analysis with the indicated antibodies (right) of Kms18 MM cells untreated or treated with 5 nM BTZ for 48 hours. qRT-PCR data are presented as the mean ± SD of 3 independent experiments, with values normalized to actin expression. Error bars represent the standard error of 3 separate experiments. Student t test (∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001). (B) Schematic representation of the experimental setup for detecting NRF1 transcript levels in NDMM samples treated and untreated with BTZ. PCs (CD138+) isolated from a cohort of 9 patients with NDMM were maintained in culture for 24 hours and then untreated or treated with 5 nM BTZ for 24 hours. (C) qRT-PCR analysis of NRF1 expression levels in the cohort described in panel B. Values were normalized to actin expression. Student t test ∗∗∗∗P < .001 (left). Table showing the cytogenetic profiles detected at the time of the patient's diagnosis (right). (D) qRT-PCR analysis of the indicated genes in MM196 primary and resistant to BTZ (MM196-RS) cell lines. Data are presented as mean ± SD of 3 independent experiments with values normalized to actin expression. Error bars represent the SD. Student t test ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001 (left). WB analysis of TCE from primary and adapted MM196 cells to BTZ for the indicated antibodies (right). (E) Proliferation analysis at the indicated points of Kms18 MM cell transiently transfected with siControl and siNRF1 and treated or not with 5 nM BTZ. Data are presented as the mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗∗∗P < .001. (F) Kms18 MM cells transiently transfected with siControl or siNRF1 were treated with increasing BTZ concentrations. Cell viability and IC50 were measured after 48 hours. Values were calculated from the fitted curves. Data are presented as the mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗∗P < .005. Bar plot (middle) showing IC50 levels obtained in the figure on the left. WB analysis (right) of NRF1 protein levels upon siNRF1. (G) Proliferation assay of primary MM196 and resistant MM196-RS cell lines transiently transfected with siControl or siNRF1. Data are presented as mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001. IC50, half maximal inhibitory concentration; n.s., not significant.

NRF1 upregulation mediates adaptive resistance to proteasome inhibition. (A) qRT-PCR of the indicated genes (left) and WB analysis with the indicated antibodies (right) of Kms18 MM cells untreated or treated with 5 nM BTZ for 48 hours. qRT-PCR data are presented as the mean ± SD of 3 independent experiments, with values normalized to actin expression. Error bars represent the standard error of 3 separate experiments. Student t test (∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001). (B) Schematic representation of the experimental setup for detecting NRF1 transcript levels in NDMM samples treated and untreated with BTZ. PCs (CD138+) isolated from a cohort of 9 patients with NDMM were maintained in culture for 24 hours and then untreated or treated with 5 nM BTZ for 24 hours. (C) qRT-PCR analysis of NRF1 expression levels in the cohort described in panel B. Values were normalized to actin expression. Student t test ∗∗∗∗P < .001 (left). Table showing the cytogenetic profiles detected at the time of the patient's diagnosis (right). (D) qRT-PCR analysis of the indicated genes in MM196 primary and resistant to BTZ (MM196-RS) cell lines. Data are presented as mean ± SD of 3 independent experiments with values normalized to actin expression. Error bars represent the SD. Student t test ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001 (left). WB analysis of TCE from primary and adapted MM196 cells to BTZ for the indicated antibodies (right). (E) Proliferation analysis at the indicated points of Kms18 MM cell transiently transfected with siControl and siNRF1 and treated or not with 5 nM BTZ. Data are presented as the mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗∗∗P < .001. (F) Kms18 MM cells transiently transfected with siControl or siNRF1 were treated with increasing BTZ concentrations. Cell viability and IC50 were measured after 48 hours. Values were calculated from the fitted curves. Data are presented as the mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗∗P < .005. Bar plot (middle) showing IC50 levels obtained in the figure on the left. WB analysis (right) of NRF1 protein levels upon siNRF1. (G) Proliferation assay of primary MM196 and resistant MM196-RS cell lines transiently transfected with siControl or siNRF1. Data are presented as mean ± SD of 3 independent experiments and error bars represent the SD. Student t test ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .001. IC50, half maximal inhibitory concentration; n.s., not significant.

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