Fig. 3

GNIP1 induces autophagy by recruiting LC3B and BECN1. A, B Protein expression levels of GNIP1 and LC3B in A549 cells treated with glucose- or serum-free medium as determined by western blot analysis. C, D Fluorescence microscopy was used to detect the number of autophagosomes in H1299 and A549 cells stably expressing LC3B after overexpressing GNIP1 (Olympus IX83) (left panel). Statistical analysis of the number of GFP puncta between groups under 200 × magnification. Data are presented as the average of three independent experiments (mean ± SD). *, p < 0.05 (right panel). E, F The expression level of autophagy related proteins was detected after overexpression of GNIP1 in H1299 and A549 cells. G, H Co-Immunoprecipitation assays of GNIP1 and LC3B. I Co-Immunoprecipitation assays of GNIP1 and BECN1. J Structural diagram of GNIP1 mutant. K-M LC3B and GNIP1 binding domain, immunoprecipitation assays. H1299 cells transfected with His-RBB and GFP-LC3B plasmids K, His-CBB and GFP-LC3B plasmids L or V5-BBCC and GFP-LC3B plasmids M were subjected to a Co-IP assay. N-P BECN1 and GNIP1 binding domain, immunoprecipitation assays. H1299 cells transfected with His-RBB and HA-BECN1 plasmids N, HA-BECN1 and His-CBB plasmids O or V5-BBCC and HA-BECN1 plasmids P were subjected to a Co-IP assay