Figure 4: PA stimulates Cox-2 expression on LDs.

For LD purification, 10⁹ C2C12 cells were treated with 500 μM PA or 500 μM PA plus 200 μM OA in DMEM with 10% FBS for 12 h. (A) Silver staining to assess the purity of LDs. The profile of LDs was dramatically different from the other three fractions. (B) Different cellular component marker proteins were probed byWestern blot. The LDs had little signal from other organelle marker proteins except ER, which suggests that LDs were physically associated with ER membrane. (C) LDs from different FFA-treated C2C12 cells were analyzed by Colloidal blue staining after SDS-PAGE separation. Three major bands showing differential expression were isolated for mass spectrometry analysis. (D) 95% confluent C2C12 cells were treated with ethanol vehicle, 500 μM PA, 200 μM OA,or 500 μM PA plus 200 μM OA in DMEM with 2% BSA for 12 h. Data were presented as mean ± SEM (n = 3) and compared by one-way ANOVA.**P < 0.01.(E) The expression of Cox-2 and Sqstm1 in all cellular fractions in different FFA treatments was determined using Western blot. (F) Transfected C2C12 cells were cultured on coverslips in 24-well plates. The cells were treated with ethanol vehicle, 500 μM PA, 200 μM OA, or 500 μM PA plus 200 μM OA in DMEM with 10% FBS for 12 h after24 h post-transfection and then subjected to immunofluorescence. Bar = 10 μM.