Figure 2.

TGF-β decreases the expression of TFEB in macrophages. (A) Mouse peritoneal MΦs were cultured with serum-free DMEM alone (control) or with IL-4 (15 ng/mL), IL-10 (20 ng/mL), m-CSF (25 ng/mL), IL-6 (20 ng/mL), TGF-β (10 ng/mL), or EO771 tumor-conditioned medium for 24 h. TFEB expression was analyzed by qPCR. *p < 0.05 vs. control. (B) Western blot assay of TFEB in MΦs treated as in (A). (C) TGF-β concentrations in indicated media were measured by ELISA. (D) Mouse peritoneal MΦs were treated with TGF-β at various concentrations for 24 h. TFEB expression was analyzed by qPCR. *p < 0.05 vs. control (0 ng/mL TGF-β). (E) Mouse peritoneal MΦs were treated with TGF-β (10 ng/mL) or EO771 TCM in the presence or absence of TGF-β-neutralizing antibody (20 μg/mL) for 24 h, TFEB expression was analyzed by qPCR. *p < 0.05 vs. DMEM. (F) Western blot analysis of TFEB protein in MΦs treated as in (E). (G) Western blot analysis of TFEB protein levels in cytosolic or nuclear subcellular fractions of MΦs treated with IL-4 (15 ng/mL), TGF-β (10 ng/mL), or EO771 TCM. TATA-box-binding protein (TBP) and actin represent control proteins for the nuclear and cytosolic fraction, respectively. Quantification of relative intensity of the protein bands is shown under the lanes. (H) TFEB expression in mouse peritoneal MΦs treated with PD98059 (25 μM), LY294002 (40 μM), and EO771 TCM for 24 h. *p < 0.05 vs. DMEM. (I) Western blot analysis of cell lysates of peritoneal MΦs treated as in (H).