Figure 5.

ABA and PPARγ signaling pathway induces PCa cellular dormancy in the bone marrow microenvironment. (A) ABA production was identified in culture media of MC3TC-E1 cells at 72 h as quantified by ELISA (cat. LS- F4483-1, Life Span Biosciences). ABA production was normalized to total protein. (B) % of G_0, G_1, or S/G₂/M cell cycle phase when PC3^VC-Control or PC3^VC-shPPARγ cells are co-cultured with murine osteoblasts (MC3T3-E1 cells) with or without ABA (50 µM) treatment. Live cells (cat. NBP2-31156, DAPI, NOVUS) were negatively gated for anti-mouse H-2kd (cat no. 116622, PE/Cy7, BioLegend), which were then positively gated for (human) HLA-A,B,C (cat no. 311426, APC/Cy7, BioLegend). After these gates were applied, the cells were plotted on the Venus-Cherry spectrum. Cell cycle phase was determined using FACS analyses. (C) Diagram of the experimental procedures for in vivo animal model. PC3^VC-Control or PC3^VC-shPPARγ cells (2 × 10⁵ cells) were suspended in 30μl of PBS and injected into 5- to 7-wk-old male CB.17. SCID mice by i.t. injection. After PCa cell injection, vehicle or ABA (20 mg/kg) treatment for 8 times (twice daily) was followed by i.p. injection. At 4 d, mice were sacrificed, and the tibiae which PCa cells were injected were collected for analyzing cell cycle phase of PC3^VC cells by FACS analyses. Antibody staining were applied as same as in coculture study in A. (D) Quantification of % of G_0, G_1, or S/G₂/M cell cycle phase from 5C (n = 4/group). Data in A, B and D are representative of mean with SD (Student's t test).