Figure 2: Human TRIM31 induces autophagy.

(a) Starvation enhances colocalization of endogenous TRIM31 and LC3 in the intestinal epithelial cell line, Caco-2. Cells were incubated with complete media (-starvation) or serum-free DMEM for 6 h, immunostained with anti-TRIM31 and anti-LC3 antibodies, and then analysed by confocal microscopy. Scale bars, 5 μm. (b) TRIM31-Myc (red) colocalizes with GFP-LC3 (green) and accumulates LC3-positive puncta in HeLa cells. TRIM31-Myc was stained using anti-Myc antibody and analysed by IFA. Blue, DAPI. Scale bars, 5 μm. Right graph, quantification of LC3 puncta in HeLa cells expressing either Mock or TRIM31-Myc. *P<0.01 (Student’s t-test). (c) Overexpressed TRIM31-Myc upregulates LC3-II in HeLa (upper panels) and HEK293T (bottom panels) cells. HeLa cells were starved in serum-free DMEM for 8 h and then subjected to immunoblot analysis with the indicated antibodies. (d) The LC3-II band intensities in c were quantified by scanning densitometry and normalized to the β-actin or tubulin band intensity. *P<0.01 (Student’s t-test). (e) Starvation leads to the enrichment of autophagosomal components in the pellet fraction of HeLa cells. HeLa cells were transfected with plasmids expressing TRIM31-Myc, Myc-Atg7, HA-Atg12, Atg5-HA or EGFP-LC3 and then starved in serum-free DMEM for the indicated times. Cells were separated into soluble and pellet fractions by centrifugation, and then subjected to immunoblot analysis with the indicated antibodies. TOM20 and Tubulin were used to indicate the mitochondrial and cytoplasmic compartments, respectively. (f) TRIM31-Myc-expressing HeLa cells were starved in serum-free DMEM and then incubated with chloroquine. Cells were lysed with 1% NP-40 and separated into soluble and pellet fractions by centrifugation. Samples were subjected to SDS–PAGE and detected with primary antibodies as indicated. All data are representative of at least two independent experiments (mean±s.d. in b,d).