Fig 2: High HJURP and YAP1 expression in TNBC cells.A Western blot analysis of HJURP and YAP1 expression in breast cancer cell lines (MCF-7, T47D, ZR-75-1, SK-BR-3, MDA-MB-231, BT549, HS578T, HCC1937, and BCAP-37). B, C Oncomine database showed the expression of HJURP and YAP1 in normal breast tissues, triple-negative breast cancer, and non-triple-negative breast cancer. D Kaplan–Meier analyses RFS based on HJURP and YAP1 mRNA levels, using the KM-plotter breast cancer database (http://kmplot.com/analysis/). A median cutoff was chosen in the analysis. E, F The expression of HJURP in MAD-MB-231 cells and BT549 cells stably transfected with or without HJURP shRNAs was explored by western blot analysis and RT-PCR analysis. Data were presented as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ns not significant.

Fig 3: HJURP/YAP1 axis induces NDRG1 transcription.A, B Western blot analysis and RT-PCR analysis of NDRG1 expression in MDA-MB-231 cells and BT549 cells stably expressing Sh-NC or Sh-YAP1. C Enrichment of YAP1 in the NDRG1 promoters was measured by ChIP experiments in MDA-MB-231 cells and BT549 cells. D Promoter activities of NDRG1 in MDA-MB-231 cells and BT549 cells stably expressing Sh-NC or Sh-YAP1. E, F Different promoter activities of NDRG1 in 293 T transfected with Flag vector or Flag-YAP1, as measured by a dual-luciferase assay. G, H MDA-MB-231 cells and BT549 cells stably expressing Sh-NC or Sh-YAP1 were transfected with the Flag vector or Flag-NDRG1 and then seeded. Cell proliferation was then determined by the CCK-8 assay at 24, 48, and 72 h, respectively. Data were presented as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ns not significant.

Fig 4: HJURP inhibits YAP1 ubiquitination.A Western blot analysis of YAP1 expression in MDA-MB-231 cells and BT549 cells with or without HJURP depletion in presence of proteasome inhibitor MG-132 (10 μM, 6 h) or lysosome inhibitor chloroquine (10 μM, 24 h). B Endogenous HJURP was analyzed by immunoprecipitation with YAP1 antibody in MDA-MB-231 cells and BT549 cells. C MDA-MB-231 cells and BT549 cells stably expressing Sh-HJURP were transfected with HA-Ub plasmid 48 h. Ubiquitination of YAP1 was analyzed by immunoprecipitation with YAP1 antibody and confirmed by western blot analysis. D, E Cell proliferation of MDA-MB-231 cells and BT549 cells stably expressing Sh-NC or Sh-HJURP was then determined by the CCK-8 assay at 24,48, and 72 h, respectively. F, G MDA-MB-231 cells and BT549 cells stably expressing Sh-NC or Sh-HJURP were transfected with the Flag vector or Flag-YAP1 and then seeded. Cell proliferation was then determined by the CCK-8 assay at 24,48, and 72 h, respectively. Data were presented as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ns not significant.

Fig 5: HJURP/YAP1 axis regulates NDRG1 transcriptional level.A, B RNA sequence analysis was performed in MDA-MB-231 cells stably expressed Sh-NC or Sh-YAP1 (n = 3). It showed differential transcriptomic expression. C The Heatmap diagram with transcripts RNAs in MDA-MB-231 cells stably expressing Sh-NC or Sh-YAP1 (n = 3). The color scale at the right illustrates the relative expression level of each transcript across all samples; red and blue are expression levels above and below the mean, respectively. White is the mean expression level. D Top changed pathways in Sh-YAP1 groups versus Sh-NC groups were shown. E Kaplan–Meier analyses RFS based on YAP1 and NDRG1 mRNA levels, using the KM-plotter breast cancer database (http://kmplot.com/analysis/). A median cutoff was chosen in the analysis.