Fa free bovine serum albumin bsa

Mφs were cultured in control RPMI 1640 supplemented with 10% FBS, 11 mM GLC, and 2 mM GLN or in modified medium supplemented with altered nutrients, including FBS, GLC, AAs, and FAs. In brief, a stock solution of each nutrient was prepared and subsequently added to cells, after which the final concentration of each nutrient was considered lower (2.5%, v/v) FBS or higher (20%, v/v) FBS, higher concentrations of GLC (20 mM, Sigma-Aldrich), higher concentrations of GLN (8 mM, GlutaMAX, Gibco), BCAAs (5 mM), or OA (100 μM, Aladdin, Shanghai, China) dissolved in FA-free bovine serum albumin (BSA; Solarbio, Beijing, China). The BCAA stock solution was prepared as a mixture of leucine, isoleucine, and valine at 100 mM (Sigma-Aldrich), and BSA alone was used as a control in the OA supplementation experiments. In concentration course tests of GLC or GLN, cells were cultured in GLC-free RPMI (11879020, Gibco) supplemented with GLC (0, 2, and 10 mM; A2494001, Gibco) or GLN-free RPMI supplemented with GLN (0, 2, and 10 mM) for 24 hours. In the case of GLN-depleted conditions, cells were cultured in GLN-free RPMI 1640 (21870076, Gibco) supplemented with (GLN⁺, 2 mM) or without GLN for 24 hours (GLN⁻). After incubation for the indicated times, the cells or conditioned medium from each group was collected for further tests.

Antibodies against cleaved caspase-3 (9679S, 1:800), p62 (39749S, 1:800), JNK (9252S, 1:1,000), phospho-JNK (4668S, 1:800), p38MAPK (8690S, 1:1,000), phospho-p38 MAPK (4511S, 1:800), β-Actin (8457S, 1:3,000) and GAPDH (5174S, 1:3,000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies for LC3B-II were purchased from Abcam (ab51520, 1:3,000; Cambridge, MA, USA). Fetal bovine serum (FBS) was obtained from Gemini Bio Products (900108; Woodland, CA, USA). FA-free bovine serum albumin (BSA) was obtained from Solarbio (A8850; Beijing, China). PA (P0500), SP600125 (S5567), and SB203580 (S8307) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rapamycin (RA, S1039) and 3-methyladenine (3-MA, S2767) were obtained from Selleck Chemicals (Houston, TX, USA).