Fig. 4

Specific interaction between ZBTB16 and G6PD in the combination treatment of pyrotinib plus chrysin. a Heat map analysis of differentially expressed mRNA in breast cancer tissues and adjacent tissues in the TCGA database. b Ubibrowser database predicting the E3 ubiquitin ligases that may be involved in the regulation of G6PD ubiquitination. c Volcano map analysis of differentially expressed mRNA in breast cancer tissues and adjacent tissues in the TCGA database. d Venn diagrams analysis of the intersection genes. e RT-qPCR detection of the mRNA expression of PML and ZBTB16 after drug combination treatment (n = 3). f Western blot detection of the protein expression of ZBTB16 after combined drug treatment (n = 3). g Interaction analysis between ZBTB16 and G6PD through co-immunoprecipitation experiment. h–j The GEPIA database determining ZBTB16 expression level in the TCGA database samples with different tumor stages. k Relevance of the prognosis of patients with ZBTB16 levels determined by the Kaplan–Meier plotter. l The GEPIA database determining the correlation between G6PD and ZBTB16 expression level. m Western blot analysis of G6PD protein half-life in SK-BR-3 cells with ZBTB16 silence. Cells were co-incubated with cycloheximide (CHX, 50 μg/ml) for 0, 4, 8, and 12 h. n Determination of the ubiquitination of G6PD in cells pretreated with 10 μM MG-132 for 3 h. Cells were transfected with ubiquitin after different treatments. The ubiquitinated G6PD was subjected to immunoprecipitation before western blot with ubiquitin antibody. All bar values are represented as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001