Fig. 4.

ATRA cooperates with ATO to induce Pin1 degradation and inhibit cancer cell growth by increasing cellular ATO uptake via the induction of AQP9 expression. a–c Correlation between the ability of ATO to induce Pin1 degradation and to inhibit cell growth. Ten human breast cancer cells were treated with ATO for 3 days, followed by Pin1 immunoblot (a) and counting cell numbers (b), and determining their correlation (c). d–f Correlation between AQP9 expression and the ability of ATO to degrade Pin1 and to inhibit cell growth. AQP9 expression were assayed by immunoblot (f) and their correlations with the ability of ATO to degrade Pin1 (d) and inhibit cell growth (e) were calculated using the data from a, b, and f. g–i AQP9 KD reduces ATO sensitivity in ATO-sensitive cells. Stable AQP9 KD 231 cells generated using two unrelated shRNA vectors were treated with different concentrations of ATO for 3 days, followed by assaying Pin1 levels (g) and cell growth (h), or with 1 µM ATO for different times, followed by assaying cellular ATO concentrations by ICP-Mass Spec (i). j–l AQP9 OX reverses ATO resistance in ATO-resistant cells. Stable AQP9 OX MCF7 cells were treated with different concentrations of ATO for 3 days, followed by assaying Pin1 levels (j), cell growth (k), or with 1 µM ATO for different times, followed by assaying cellular ATO concentrations by ICP-Mass Spec (l). m–p ATRA induces AQP9 protein expression, increases ATO uptake, and cooperates with ATO in inhibiting cell growth in TNBC cells. The 231 cells were treated with different concentrations of ATRA for 7 days, followed by AQP9 immunoblot (m) or with 1 µM ATO for different times before subjecting to ATO concentrations by ICP-Mass Spec (n), or with different concentrations of ATO and/or ATRA for 3 days, followed by counting cell number (o) and determining their synergy using CalcuSyn (p). q–s AQP9 KD abolishes ATRA cooperation with ATO, but does not affect ATRA sensitivity. Control or AQP9 KD 231 cells were treated with ATO and/or ATRA, followed by Pin1 immunoblot (q) and cell growth assay (r), followed by using CalcuSyn to calculate their synergy (s). The results are expressed as mean ± SD and the P values were determined by ANOVA test