Fig. 6: TRIM21 inhibits OS cell differentiation in an autophagy-dependent manner.

A–D OS cells were transfected with HA-TRIM21 (A) or siRNA of TRIM21 (C) and performed western blotting assay. The corresponding quantitative analyses of the RUNX2/GAPDH ratio were shown in B and D (n = 3, SEM). E U2-OS cells were transfected with siRNA of TRIM21 and harvested for detection of ALP activity using ELISA assay (n = 3, SEM). U2-OS cells were transfected with HA-TRIM21 (F) or TRIM21 siRNA (G), respectively, and collected for detection of the expression of TRIM21, ALP, and RUNX2 by qRT-PCR assay (n = 3, SEM). H U2-OS cells were transfected with HA-TRIM21 and treated with CQ and MG132 (100 nM) for 24 h. The cells were harvested and performed western blotting assay with the indicated antibodies. The ratio of RUNX2/GAPDH was shown in its lower panel (n = 3, SEM). I U2-OS cells were transfected with HA-TRIM21 or HA-vector and harvested for detection of ALP activity using ELISA assay (n = 3, SEM). J A model of the relationship between TRIM21 and differentiation. TRIM21 OE: TRIM21 overexpression. K OS cells transfected with si-NC or si-ANXA2 were performed western blotting assay (Upper panel). RUNX2/GAPDH was shown in its lower panel (n = 3, SEM). L U2-OS cells were transfected with EGFP-V or EGFP-ANXA2 and treated with RAPA (1.5 μM, 24 h) as indicated to perform western blotting assay (upper panel). Lower panel: the ratio of RUNX2/GAPDH (n = 3, SEM). M U2-OS cells stably expressing H125-TRIM21 or H125-V were transfected with EGFP-ANXA2 or EGFP-vector and treated with TET as indicated to perform western blotting assay. RUNX2/β-actin was shown in its lower panel (n = 3, SEM). *p < 0.05; **p < 0.01. All data were representative of three independent experiments.