Fig 2: PPAR? was retained in the cytosol upon MEK1-dependent mechanisms. (A) Total proteins were extracted and MEK1/2 and their p-MEK1 detected by Western blot in MCF-7 cells, MDA-MB-231 cells, MCF-7 cells transfected with Myc-empty or Myc-Skp2; (B) Protein expression was normalized against MEK1/2. Statistical differences compared with the controls were given as *p < 0.05; (C) MDA-MB-231 cells were treated with 10 µM of MEK inhibitor PD98059 or U0126 for 12 h, and the cells were subjected to cellular fractionation and blotted with PPAR? antibodies. Control cells were incubated with DMSO vehicle for the same period of time; (D) The blot for PPAR? was quantified by densitometry and the expression levels relative to Lamin B or Tubulin were calculated. The data are means ± SEM (n = 3, *p < 0.05, compared with control: Basal); (E) MDA-MB-231 cells were treated with 10 µM of MEK inhibitor PD98059 or U0126 for 12 h, and the cells were fixed and stained with PPAR? antibodies or Hoechst, and the localization was assessed by fluorescence microscopy as above.

Fig 3: Cytosolic retention of PPAR? upon overexpression of Myc-Skp2. (A) MCF-7 cells were transiently transfected with Myc-Skp2 or Myc-empty. Forty-eight hours after transfection, the cells were subjected to cellular fractionation and analyzed by Western blot to detect PPAR? localization; (B) Blot for PPAR? was quantified by densitometry and expression level relative to Lamin B or Tubulin were calculated. The data were means ± SEM (n = 3, *p < 0.05, compared with Myc-empty: PCDNA); (C) MCF-7 cells were grown on coverslips and transiently transfected with either Myc-empty or Myc-Skp2. Forty-eight hours after transfection, the cells were fixed and stained with PPAR? antibodies or Hoechst, and the localization was assessed by fluorescence microscopy as above.

Fig 4: Representative immunohistochemistry slides of Skp2 and PPAR? staining in locally advanced breast cancer. PPAR? and Skp2 protein expression in paraffin-embedded human breast benign and malignant tumors (×400). (A–C) PPAR? reactivity; (D–F) Corresponding Skp2 staining. (A) Benign breast disease sample displaying strong nuclear staining for PPAR?; (B) Infiltrating ductal carcinoma (ER-negative, PR-negative, and HER-2-negative, Grade III) showing strong cytoplasmic PPAR? immunostaining; (C) ER-positive human breast cancer sample (Grade I) showed reducing cytoplasmic PPAR? staining; (D) Benign breast disease sample displaying weak Skp2 immunostaining; (E) Infiltrating ductal carcinoma (ER-negative, PR-negative, and HER-2-negative, Grade III) showing strong Skp2 staining; (F) ER-positive human breast cancer sample (Grade I) showed weak Skp2 abundance.

Fig 5: FNIP interacts with SCFß-TRCPA. Immunoblot (IB) analysis of whole cell lysates (WCL) and Flag-immunoprecipitates (IP) derived from 293T cells transfected with Flag-FNIP2 and empty vector (EV), Myc-Cullin1, 2, 3, 4A, or 5, as indicated. At 24 h post transfection, cells were treated with 15 µM MG132 for 12 h before harvesting. B-C. IB analysis of WCL and Flag-IP derived from 293T cells transfected with Flag-FNIP2 and Myc-SKP1 (B) or Myc-RBX1 (C) as indicated. At 24 h post transfection, cells were treated with 15 µM MG132 for 12 h before harvesting. D. IB analysis of WCL derived from HeLa cells infected with lentiviral shRNA constructs against GFP and Cullin1 (four independent shRNAs namely Cullin1-A, B, C, or D) followed by selection with 1 µg/mL puromycin for 72 h to eliminate non-infected cells. E. IB analysis of WCL derived from HeLa cells infected with lentiviral shRNA constructs against GFP, ß-TRCP1, ß-TRCP2, ß-TRCP1+2 (shRNA against both ß-TRCP1 and ß-TRCP2 paralogs), and SKP2 (three independent shRNAs namely SKP2-A, B, and C) followed by selection with 1 µg/mL puromycin for 72 h to eliminate non-infected cells. F. IB analysis of WCL and HA-IP derived from HeLa cells transfected with HA-FNIP2 along with empty vector (EV), Flag-ß-TRCP1, or Flag-SKP2. At 24 h post transfection, cells were treated with 15 µM MG132 for 12 h before harvesting. G. IB analysis of WCL and FNIP2-IP derived from HeLa cells treated with MG132 for 12 h before harvesting. Endogenous interaction between FNIP2 and ß-TRCP1 was confirmed. Input, 5% WCL. H. IB analysis of WCL and Flag-IP derived from HeLa cells transfected with Flag-FNIP2 along with empty vector (EV), Myc-ß-TRCP1, or Myc-ß-TRCP1-R474A. At 24 h post transfection, cells were treated with 15 µM MG132 for 12 h before harvesting.