Crystalline silica (CS) induces transcription factor EB (TFEB) nuclear localization and increases TFEB expression in vivo. (A,B) Immunoblotting analysis of TFEB levels in the cytosol or nucleus of lung tissue on day 7 or day 56 post CS instillation; β-actin, loading control for cytosolic proteins; and lamin B, loading control for nuclear proteins (n = 4). (C) qPCR analysis of TFEB expression in lung tissue on day 7 or day 56 (n = 5 to 6). (D) Immunofluorescence analysis of the distribution of F4/80 and TFEB in lung tissue on day 7 or 56 post saline or CS treatment. Red arrows indicate alveolar macrophages (AMs) in the lungs containing silicotic lesions with predominantly nuclear TFEB staining (scale bar = 50 μm and n = 5). (E) Immunofluorescence analysis of TFEB nucleus translocation in primary AMs from bronchoalveolar lavage fluid (BALF) (scale bar = 10 μm and n = 3 to 4). (F) qPCR analysis of TFEB expression in primary AMs on day 7 or 56 post CS instillation (n = 4). Data are presented as the mean ± SD. *, p < 0.05; **, p < 0.01; and ##, p < 0.01.