Moderate reduction in PTEN decreases endocrine sensitivity in ER+/HER2- breast cancer cell models. MCF7L-shPTEN cells with two different shRNA sequences (#1 and #2) (A and B), T47D-shPTEN (#1) cells (C), and BT483-shPTEN (#1) cells (D) were grown in PRF medium with 5% CS-FBS under -/+Dox for three days, then treated with E2 (1 nM) as control, continuing the same medium (ED), Tam (100 nM), or Ful (100 nM) for five days in 96-well plates. Cell growth (%) at day 5 in all anti-estrogen groups (ED, Tam, and Ful) was normalized to E2 -/+Dox control. There is no noticeable change in cell growth between E2-Dox and E2 + Dox groups. Cell growth was monitored daily by in situ cell cytometry (Celigo). (E and F) MCF7L-shPTEN and T47D-shPTEN cells were prepared as before with an additional range of Dox induction and subjected to endocrine treatment. Cell growth (%) at day 5 in all anti-estrogen groups (ED, Tam, and Ful) was normalized to E2 -/+Dox control. The Bonferroni post hoc test was used for all pairwise comparisons (*P <0.05, **P <0.01, ***P <0.001). CS-FBS, charcoal stripped-fetal bovine serum; Dox, doxycycline; E2, β-estradiol; ED, estrogen deprivation; ER, estrogen receptor α; Ful, fulvestrant; HER2, human epidermal growth factor receptor 2; PRF, phenol-red free; PTEN, phosphatase and tensin homolog; shRNA, short hairpin RNA; Tam, tamoxifen.