Fig. 4: PPP3CB-mediated decrease of p-FOXO3a levels altered the feedback regulation.

a, b SKBR3 or BT474 cells were treated with vehicle (Mock) or Rapamycin (10 μM, Rapa) in combination with rhIGF2 at indicated concentrations for 24 h. The expression of p-Akt^(S473), Akt, p-S6K, S6K, p-FOXO3a, FOXO3a, IRS1, PPP3CB, and β-actin was examined by western blot assays (a); The expression levels of miR-128-3p and miR-30a-5p were detected by qRT-PCR (b), ****p < 0.0001. c SKBR3 or BT474 cells were treated with a series of phosphatase inhibitors as Cantharidic acid (0.5 μM), Endothall (1 μM), RK-682 (10 μM), Cypermethrin (1 μM), Deltamethrin (1 μM), RWJ-60475 (2 μM), Tyrphostin 8 (10 μM), CinnGel (1 μM), BML-260 (10 μM), or BN-82002 (5 μM) in combination with rhIGF2 at indicated concentrations for 24 h. The expression of p-FOXO3a, FOXO3a, and β-actin was analyzed by western blot assays. d SKBR3 cells were treated with rhIGF2 at indicated concentrations for 24 h. The expression of PPP3CB, PPP3CA, PPP3CC, PPP3R1, PPP3R2, and β-actin was examined by western blot assays. e, f SKBR3 or BT474 cells with specific knockdown of PPP3CB by shRNAs (sh-Con vs sh-1# and sh-2#) were treated with rhIGF2 (80 ng/ml) for 24 h. The expression of PPP3CB, p-FOXO3a, FOXO3a and β-actin were examined by western blot assays (e); The enrichment of FOXO3a at the promoters of miR-128-3p and miR-30a-5p were detected by ChIP-qPCR (f), ****p < 0.0001. n = 3 biological independent samples (b, f). Data are presented as mean values ± SEM (b, f). Statistical significance was determined by a two-tailed Student’s t-test (b, f). Data show a representative of three independent experiments (a, c, d, e). All data are provided in the Source Data.