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. 2018 Jul 18;51(2):576–592. doi: 10.4143/crt.2018.100

Fig. 3.

Fig. 3.

Paired-like homeodomain transcription factor 2 (PITX2) inhibition sensitizes MCF7/LR cells to letrozole treatment. (A) MCF7/LR cells were transfected with PITX2 shRNA or scramble shRNA, followed by puromycin selection for 1-2 weeks. Stable knockdown of PITX2 expression was then verified by Western blotting analysis. Actin served as the loading control. (B) After a 3-day culture and a following overnight starvation, MCF7/LR cells stably transfected with PITX2 shRNA or scramble shRNA were treated with 10–5 M of letrozole, along with 25 nM of androstenedione, for another 4 days. Viable cell numbers were then determined using trypan blue staining. Fold change was determined for each treatment relative to the untreated control cells. (C) After a 3-day culture and a following overnight starvation, MCF7 cells stably transfected with pPM-His-PITX2 or pPM-His vector were treated with 10–5 M of letrozole, along with 25 nM of androstenedione, for another 4 days. Cell apoptosis was determined using an apoptosis enzyme-linked immunosorbent assay kit. (D) Tumor xenograft assay, as described above, was carried out to evaluate the effects of PITX2 inhibition on in vivo letrozole sensitivity. *p < 0.05 and **p < 0.01 when comparing letrozole (10 μg/day)+scramble shRNA to letrozole (10 μg/day)+PITX2 shRNA. (E) Cell invasiveness assay as described above. Shown are the absorptions at 570 nm of MCF7/LR cells treated with different transfection plus 10–5 M of letrozole for 48 hours, in the lower chambers of transwells.