Ibmx m

3T3-L1 murine white preadipocytes (ATCC, Manassas, VA, USA) were grown up to the contact inhibition stage and remained in the post-confluent stage for 2 days in DMEM supplemented with 10% calf bovine serum (Gibco, Gaithersburg, MD, USA) and penicillin-streptomycin (Welgene, Daegu, Korea). Differentiation was induced by changing the medium to DMEM supplemented with 10% FBS (Welgene, Daegu, Korea) plus a cocktail of hormones (MDI) that include 0.5 mM IBMX (M) (Sigma, St. Louis, MO, USA), 0.5 µM dexamethasone (D) (Sigma), and 5 µg/mL insulin (I) (Sigma, St. Louis, MO, USA) in the absence or presence of CTXA at the indicated concentrations. In this study, the cell culture medium containing CTXA was vigorously vortexed for 30 s before addition to cells. After 48 h MDI-induction, the differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 µg/mL I in the absence or presence of CTXA at the designated doses for additional 3 days. The cells were then fed every other day with DMEM containing 10% FBS in the absence or presence of CTXA at the indicated concentrations for additional 3 days.

3T3-L1 preadipocytes (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene, Daegu, Korea) supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (Welgene, Daegu, Korea) at 37 °C in a humidified atmosphere of 5% CO₂. 3T3-L1 preadipocytes were grown up to the contact inhibition stage and remained in the post-confluent stage for 2 days (D0). Differentiation was induced by changing the medium to DMEM supplemented with 10% FBS (Welgene, Daegu, Korea) plus a cocktail of hormones (MDI) that include 0.5 mM IBMX (M) (Sigma), 0.5 µM dexamethasone (D) (Sigma), and 5 µg/mL insulin (I) (Sigma) for 2 days (D2). Cells were switched to DMEM supplemented with 10% FBS and 5 µg/mL insulin for additional 3 days (D5). Cells were then fed every other day with DMEM supplemented with 10% FBS for further 3 days (D8).

3T3-L1 murine white preadipocytes (ATCC, Manassas, VA, USA) were grown up to the contact inhibition stage and remained in the post-confluent stage for 2 days in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% calf bovine serum (Gibco, Gaithersburg, MD, USA) and penicillin-streptomycin (Welgene, Daegu, Korea). Differentiation was then induced by changing the medium to DMEM supplemented with 10% FBS (Welgene) plus a cocktail of hormones (MDI) that include 0.5 mM IBMX (M) (Sigma, St. Louis, MO, USA), 0.5 μM dexamethasone (D) (Sigma) and 5 μg/mL insulin (I) (Sigma) in the presence or absence of tanshinone IIA at the indicated concentrations. After 48 h MDI-induction, the differentiation medium was replaced with DMEM supplemented with 10% FBS and 5 μg/mL insulin in the presence or absence of tanshinone IIA at the indicated concentrations. The cells were then fed every other day with DMEM containing 10% FBS in the presence or absence of tanshinone IIA at the indicated concentrations until day eight. On day eight, the preadipocytes became mature adipocytes that rounded-up and filled with many oil droplets.