Fig. 1

Identification of USP36 as a novel mediator of ER signaling in ER positive breast cancer. A The flowchart shows the siRNA screening procedure for identifying novel deubiquitinases involved in modulating ER signaling. Each of the 76 human DUB genes was knocked down in MCF-7 cells with 20 μM pooled siRNAs. After 48 h, the quantitative gene expression analysis was detected by real-time PCR. B The relative expression level of TFF1(PS2) in MCF-7 cells transfected with DUBs in the screening library. The real-time PCR results of siControl is normalized to 1. C The expression of USP36 in breast cancer tissues (n = 1097) and normal tissues (n = 114) from TCGA database (https://www.genome.gov/). D The expression of USP36 in ER positive breast cancer tissues (n = 566) and normal tissues (n = 114) from TCGA database (https://www.genome.gov/). E–F Kaplan − Meier analysis showing relapse-free survival depending on USP36 expression levels in ER positive and ER negative breast cancer from public meta-analysis data (https://kmplot.com). G-H Immunohistochemistry (IHC) detecting USP36 expression in 65 breast cancer samples and 55 normal breast tissue (G). Statistical analysis of USP36 expression in G (H). I Volcano map of RNA-seq data from MCF-7 cell lines treated with siControl or siUSP36. |log2Fold change|> 1 and P value < 0.05 are set as screening criteria. J KEGG analysis of downregulated genes in RNA-seq data from MCF-7 cell lines treated with siControl or siUSP36 with threshold criteria of P < 0.05 and fold change > 1.5. K Gene set enrichment analysis (GSEA) shows enrichment of estrogen response genes in RNA-seq data from MCF-7 cell lines treated with siControl or siUSP36. L Heatmap of relationship of USP36 and differentially ER pathway related genes in RNA-seq data with threshold criteria of P < 0.05 and fold change > 1.5. M Gene set enrichment analysis (GSEA) shows enrichment of estrogen response genes in breast cancer tissue from TCGA data with threshold criteria of P < 0.05 and fold change > 1.5. All P values were calculated by unpaired two-tailed Student’s t tests. *P < 0.05; **P < 0.01; ***P < 0.001. (C-L)