Fig 2.

U2OS cells (osteosarcoma cell line; ATCC) were cultured in DMEM, 10% foetal calf serum, 100 U/ml penicillin/100 μg/ml streptomycin, 5 μg/ml plasmocin (Invivogen) at 37°C, 5% CO₂ and stably transfected GFP-WIPI-2B or GFP-WIPI-2D U2OS cell clones were selected by using 0.6 mg/ml G418 (Invitrogen). Comparable levels of expressed GFP-WIPI proteins are demonstrated by anti-GFP enhanced chemiluminescence (ECL) detection of total protein extracts (A). Protein expression of GFP-WIPI-1, -2B and -2D is shown, demonstrating relatively low expression when compared to a generated control GFP cell clone (A). Freeze fracture immuno-EM images of stable GFP-WIPI-2B (B) or GFP-WIPI-2D (C) U2OS cells upon nutrient starvation (6 hrs) identified a prominent localization of WIPI-2 in both the inner (IM) and of the outer (OM) autophagosomal membrane (AP) (B, C), and at the PM (D, E). GFP-WIPI-2B (D) and GFP-WIPI-2D (E) harbouring vesicles (P: P-face), may be premature APs, were identified close to the PM, suggesting that these vesicles might have originated from the PM. In contrast to the prominent PM localization, some WIPI-2D was also detected at the ER/NM (F) and near the Golgi cisternae (G). Antibodies: anti-GFP antiserum/ab290; Abcam; goat anti-rabbit 18 nm gold complexes; Jackson Immunoresearch. Scale bars 200 nm.