PTEN-KD enhances PI3K signaling, reduces expression of ER and its regulated genes, and promotes expression profile of genes associated with luminal B subtype. (A) Diagram of the lentiviral pINDUCER vector. (B) Fluorescence microscopy (20×) shows the eGFP and tRFP expression in MCF7L-shPTEN cells after Dox induction for 48 h. (C) The quantification of the positive population of eGFP and tRFP cells was analyzed by flow cytometry. (D) After three days of Dox induction, the cell lysates of shPTEN cell models were subjected to Western blotting. Numbers under each blot indicate protein densitometry normalized to β-actin (values in -Dox cells were set as 1). (E) Cell lysates from MCF7L-shPTEN and T47D-shPTEN cells after five days induction with a range of Dox doses were blotted by the indicated antibodies (densitometry shown under the PTEN blotting). (F) PTEN-low gene signature derived from PTEN-KD cells representing the differentially expressed genes compared to -WT cells was correlated with known gene signatures related to growth factor signaling -. Heat map of t statistic indicates the global similarity between signatures based on the Pearson’s correlations. (G) Up- or downregulated (DN) genes in PTEN-KD cells were analyzed for gene enrichment (one-sided Fisher’s exact test) in the gene sets of endocrine resistant xenograft tumors (Group 1 to 5)  or E2-induced MCF7 cells (Cluster B) . (H) Box plot shows the PTEN-low gene signature scores of each ER+ luminal tumor from datasets of TCGA  and Compendium . The mean value ± standard deviation of all samples in each subtype is marked within the box plot in red (****P <0.0001, Student’s t test). Dox, doxycycline; E2, β-estradiol; ED, estrogen deprivation; eGFP, enhanced GFP; ER, estrogen receptor α; KD, knockdown; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin homolog; TCGA, The Cancer Genome Atlas; tRFP, turbo-RFP.