Supplemental Figures S1-S8

Supplemental Figures S1-S8

Figure S1. Cip2a knockdown inhibited TNBC cell proliferation. (A) MDA-MB-231, BT549, and Hs578T were transfected with Cip2asiRNA for 48 h, cell viability was evaluated by MTS, *P <0.05. (B) MCT-7, T47D, and BT474 were transfected with Cip2a siRNA for 48 h, cell viability was evaluated by MTS. (C)MDA-MB-231, BT549, and Hs578T were transfected with Cip2a shRNAusing lentivirus,cells were selected with 2 μg/mL puromycin for 14 days. Cip2a protein expression was analyzed by using the Western blot method. -Actin was employed as an inner control. (D) MDA-MB-231and BT549were transfected with Cip2a shRNA, cell number was evaluated by cell count at the indicated times, *P <0.05.

Figure S2. Cip2a affects the p27Kip1 expression.(A) MDA-MB-231 and BT549 cells were transfected with Cip2asiRNA, the expression of p21, p27Kip1, and p18 were analyzed by Western blot. (B) MDA-MB-231 and BT549 cells were transfected with pCMV-Cip2a for 48 h, the protein expression of Cip2a, andp27Kip1 were analyzed by Western blot. (C) MDA-MB-231 and BT549 cells were transfected with pCMV-Cip2a for 48 h, the mRNAexpression of Cip2a, and p27Kip1 were analyzed by Real time RT-PCR.**P <0.01.

Figure S3. Effects of p27KIP1 siRNA on p27Kip1 expression.MDA-MB-231 and BT549 were transfected with Cip2a siRNACip2a shRNAin combination with p27Kip1 siRNA, the expression of Ci2a and p27Kip1 was confirmed by immunoblotting. β-Actin was used as a loading control.

Figure S4. Effect of Cip2a siRNA onCdk1 expression and activity.(A) MDA-MB-231 and BT549 were transfected with Cip2a siRNAfor 48 h, the expression of Cdk1, cyclin A, and cyclin B1was confirmed by western blot.(B) Left panel, a representative western blot performed with an anti-cdk1 antibody using the cell extracts prior to immunoprecipitation (INPUT). Right panel, protein kinase activity immunoprecipitated with an anti-cdk1 antibody (i.e. cdk1-specific kinase activity) from the cell extracts, normalized to the relative amount of cdk1 present in these extracts (see input in left panel). *p<0.05.

Figure S5. Effect of PP2A inhibitor on the expression and phosphorylation levels of p27Kip1 regulating by Cip2a siRNA.(A) MDA-MB-231 and BT549 were transfected with Cip2a siRNA for 48 h, PP2A activitywas measured by PP2A immunoprecipitation phosphatase assay. (B) MDA-MB-231 cells were transfected with Cip2a shRNA, and then treated with 0.25 nmol/L PP2A inhibitorO. acid for 48 h, the expression of p-p27Kip1(Ser10), and p27Kip1 were analyzed by Western blot. (C) MDA-MB-231, and BT549 were transfected with Cip2a siRNA, and then treated with 0.25 nmol/LO. acid for 48 h, cell viability was evaluated by MTS assay. *P <0.05, **P <0.01.

Figure S6.Cip2a inhibits Akt-associated PP2A activity. MDA-MB-231 cells were transfected with pCMV or pCMV-Cip2a, Control siRNAor Cip2a siRNAfor 48 h, cell lysates were immunoprecipitated by anti-Akt1 antibodies. *P<0.05.

Figure S7. Western blot analysis of p27Kip1protein levels in breast cancer cell lines.

Figure S8. Cip2a depletion sensitizes TNBC to PARP inhibition. MDA-MB-436 wastransfected with Cip2ashRNA, and then treated with PARP inhibitor AG14361 at different concentration for 48 h, cell viability was evaluated by MTS assay. Table indicates the half-maximal inhibitory concentration (IC50) values for each condition. *P <0.05.

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