Fig. 5: TRIM21 separates the ANXA2-TFEB complex and thus facilitates the autophagy in OS cells.

A U2-OS cells were used to perform co-IP assay with ANXA2 antibody. B U2-OS cells stably expressing H125-V or H125-TRIM21 were used to perform co-IP assay with IgG and ANXA2 antibodies after TET (10 μg/ml) treatment. C U2-OS cells stably expressing H125-TRIM21 were treated with DMSO or TET and then performed IF using ANXA2 and TFEB antibodies (Upper panel). (Lower panel) Percentage of the cells with colocalization of ANXA2 and TFEB in 50 cells. D U2-OS cells were transfected with si-NC or si-ANXA2 and then performed IF using ANXA2 and TFEB antibodies (upper panel). Lower panel: percentage of the cells with nuclear localization of TFEB in 80 cells. Arrows: nucleus. E U2-OS cells stably expressing H125-TRIM21 or H125-V were treated with TET and then performed IF using the TFEB antibody (Upper panel). Lower panel: Percentage of the cells with nuclear localization of TFEB in 80 cells. Arrow: nucleus. U2-OS cells were transfected with si-ANXA2 (F) or si-TRIM21 (G) and then performed subcellular fractionation assay to isolate the nuclear and cytoplasmic fractions (Upper panel). The corresponding quantitative analyses of TFEB/References were shown in their lower panels, respectively (n = 3, SEM). H, I U2-OS cells transfected with si-ANXA2, EGFP-ANXA2 or si-TRIM21, or the stable U2-OS cells expressing H125-TRIM21 or H125-V with TET treatment as indicated to perform qRT-PCR assays to analyze the mRNA expression of p62, ANXA2, or TRIM21 (n = 3, SEM). J U2-OS cells stably expressing H125-TRIM21 or H125-V were transfected with EGFP-ANXA2 as indicated and treated with TET to perform the subcellular fractionation assay using the indicated antibodies. K The quantitative analyses of TFEB/References in the nucleus and cytoplasm in J (n = 3, SEM). L A supposed association of TRIM21, ANXA2, and TFEB. Scale bar: 10 μm. *p < 0.05, **p < 0.01, ***p < 0.001. All data were representative of three independent experiments.