Fig 1: ABA and PPAR? 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 G0, G1, or S/G2/M cell cycle phase when PC3VC-Control or PC3VC-shPPAR? 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. PC3VC-Control or PC3VC-shPPAR? 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 PC3VC cells by FACS analyses. Antibody staining were applied as same as in coculture study in A. (D) Quantification of % of G0, G1, 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).