Nupr1 is a key mediator of chemoresistance in L-DOXR cells
(A) Volcano plot of differential gene expression between WT and L-DOXR cells. Cut-off criteria included a fold change of 2. (B, C) Ingenuity Pathway Analysis (IPA) of the RNA-sequencing data shows disease and disorders (left), causal network (middle), canonical pathways (Yue et al., 2020), and upstream regulator (C). The top five ranks are presented. Cut-off criteria are p < 0.05 and a false discovery rate (FDR) q-value < 0.05. (D) Kaplan-Meier (Dai et al., 2013) survival curve represents the overall survival rate in chemotherapy-treated TNBC patients (n = 66) based on low vs. high Nupr1 expression from the meta-analysis in KM plotter. (E) KM survival curve representing the relapse-free survival rate in chemotherapy-treated patients (n = 135) based on low vs. high Nupr1 expression from GSE12093. (F) Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis of Nupr1 mRNA expression in cells (left) and tumor tissue from a mouse xenograft (Yue et al., 2020). The values were normalized to the level of the control (Jazaeri et al., 2005). (G) The relative luciferase activity of the Nupr1 promoter was measured in WT cells and L-DOXR cells. (H) Cell viability was measured among si-Nupr1 transfected cells treated with DOX using a crystal violet staining assay. (I) Cell viability was measured after administering DOX with and without ZZW-115 (Nupr1 inhibitor). The bar graph indicates the average density of dyed crystal violet. (J) Apoptotic proteins were detected by immunoblotting from WT cells and L-DOXR cells with and without DOX/ZZW-115. (K) Timeline demonstrating the subcutaneous injection of 1 × 107 L-DOXR cells followed by injections of doxorubicin (4 mg/kg) or ZZW-115 (2.5 mg/kg, 5.0 mg/kg) into the tail vein (n = 6 per group). (L) The photographs show representative tumors. Scale bar: 2 cm. (M) Representative images showing immunohistochemical staining for Nupr1 in PCGGs with and without ZZW-115 and DOX treatment (left). Localization of Nupr1 in the control and ZZW-115 (5.0 mg/kg)-injected tumors was analyzed by ImageJ (Yue et al., 2020). Scale bar: 20 μm. (N) Animals were monitored for up to 42 days, and tumor size was measured using calipers at three-day intervals. (O) Representative images showing immunohistochemical staining for TUNEL and active-caspase 3 on paraffin sections of tumor tissues. Scale bar: 20 μm. All data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; Student’s two-tailed, unpaired t-test (F); one-way ANOVA with Bonferroni’s post-test (G, H, I, N).