3 oba

Human colon cancer cell lines HT29 and RKO, murine colon cancer cell line CT26, and the human T-lymphoblastic Jurkat cell line were purchased from ATCC and conserved by our laboratory. All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA), supplemented with 100 μg/mL streptomycin (Solarbio, Beijing, China) and 100 U/mL penicillin (Solarbio, Beijing, China). Cells were cultured at 37 °C in an incubator with 5% CO₂. Metformin (purity > 97.0%, Sangon Biothech, Shanghai, China), and 3-OBA (Sigma-Aldrich, St. Louis, MO, USA) was purchased.

Enzymatically digested BM cells were seeded in eight-well Lab-Tek II Chamber slides (5 × 10⁴ cells/well, Sigma-Aldrich). Cells were cultured in α-MEM (Gibco) supplemented with 10% FBS and 1% penicillin/streptomycin (Gibco) for 3 days and then treated with sodium l-lactate (5 mM, Sigma-Aldrich), 3,5-DHBA (0.2 mM, Sigma-Aldrich) or 3-OBA (3 mM, Sigma-Aldrich). Plated MSCs were fixed with Cytofix/Cytoperm (BD) for 20 min at 4 °C and subsequently washed with Perm/Wash buffer (BD) during immunocytochemistry. After blocking with 5% BSA in PBS for 1 h, MSCs were stained with DAPI, goat anti-LepR-biotin polyclonal (R&D Systems), anti-endomucin (Abcam, V.7C7.1), anti-SDF-1α (R&D, 79018), and rabbit anti-SCF polyclonal (Abcam) antibodies overnight at 4 °C. After one wash with Perm/Wash buffer, cells were stained with DyLight^TM 488-conjugated streptavidin (BioLegend) or Alexa Fluor 546-conjugated donkey anti-rabbit IgG (Invitrogen) as secondary antibodies. All samples were viewed under a confocal laser scanning microscope (Carl Zeiss).

According to the acute results of our experiment, we found that the 0.03 M 3-hydroxy-butyrate (3-OBA, 54965, Sigma) concentration gave the best effect to activate the level of PKA phosphorylation. Based on this concentration, we examined the time effect and determined the optimal time was 15 min. Based on these results, rats received an intramuscular injection of 0.081 g/kg 3-OBA (0.03 M) in their left gastrocnemius muscle prior to exercise training at 15 min. The compound 3-OBA was dissolved in a solution of normal saline prior to being administered by injection. Control rats received an injection of an equivalent amount of saline solution.