Figure 3: TRIM31 induces LPS-induced Atg5/7-independent alternative autolysosome formation.

(a) HeLa cells expressing mRFP-GFP-TRIM31 were left untreated or treated with LPS (100 ng ml⁻¹) for 4 h. Blue, DAPI. Scale bars, 5 μm. (b) Quantification of red puncta is shown in the graph. *P<0.01 (Student’s t-test). (c,d) Wild-type or Atg5^−/− MEF cells were transfected with mRFP-GFP-LC3 or mRFP-GFP-TRIM31. After 24 h, cells were stimulated with LPS (100 ng ml⁻¹) for 10 h and analysed by IFA. Blue, DAPI. Scale bars, 5 μm. (e) Quantification of red puncta is shown in the graph. *P<0.01 (Student’s t-test). (f) Cell extracts from wild-type or Atg5^−/− cells expressing Mock or TRIM31-Myc were fractionated into pellets and supernatants by centrifugation. Detergent-insoluble pellets were resuspended in 1 × SDS loading buffer and loaded onto SDS–PAGE gels with the same amount of supernatant, and then analysed by immunoblot analysis with the indicated antibodies. Tubulin was used as a loading control. (g) HeLa cells expressing mRFP-GFP-TRIM31 and Myc-LC3 (blue) were left untreated or treated with LPS (100 ng ml⁻¹) for 4 h. Scale bar, 5 μm. (h) RING deletion mutant of TRIM31 in Atg5^−/− MEFs is present on autophagosomal (left) and autolysosomal (right) structures. Atg5^−/− cells expressing TRIM31-ΔRING-Myc were stimulated with LPS (100 ng ml⁻¹) for 3 h with chloroquine (5 μM). The localization of TRIM31-ΔRING-Myc was examined by immunogold electron microscopy using anti-Myc antibody. Arrows indicate double-membrane structure. Scale bar, 0.1 μm. (i) Atg5^−/− cells expressing TRIM31-ΔRING-Myc were left untreated or treated with LPS (100 ng ml⁻¹) for 3 h. Quantification of autophagosomal vesicles is shown in the graph. *P<0.01 (Student’s t-test). All data are representative of at least two independent experiments (mean±s.d. in b,e and i).