Fig 2: 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/ß-actin was shown in its lower panel (n = 3, SEM). *p < 0.05; **p < 0.01. All data were representative of three independent experiments.

Fig 3: Proposed cytokine signaling pathway triggered by internalized free IgG antibody. The 3D8 IgG-N434D mutant, which is not recognized by TRIM21, induces human monocytes to produce more IL-8 and TNF-a than cells exposed to wt 3D8 IgG. An as-yet-unknown cytosolic Fc sensor may be responsible for cytokine signaling triggered by entry of free IgG to the cytosol.

Fig 4: The pathway inhibitors affect the TWEAK/Fn14 regulation of Ro52 in murine keratinocytes. PAM212 cells were cultured in vitro and received ultraviolet B irradiation and TWEAK stimulation. Some cells were pretreated with the pathway inhibitors of nuclear factor-kappa B (JSH-23), mitogen-activated protein kinases/extracellular signal-regulated kinase (U0126), and phosphatidylinositide 3-kinase (wortmannin), respectively. (A) Quantitative reverse transcription polymerase chain reaction was performed for the mRNA expression levels of Ro52. (B) Accordingly, the protein expression levels of Ro52 were determined in supernatants and cell lysates. (C) The intensities of Western blotting bands were measured by using ImageJ and then normalized to the ß-actin values accordingly. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative images are shown. *p < 0.05, **p < 0.01.

Fig 5: TWEAK and Fn14 expression increases in skin lesions of cutaneous lupus erythematosus. (A) By immunohistochemistry, the expression of TWEAK and Fn14 was determined in paraffin sections. (B) The stained sections were quantitated for the positivity percentage per square millimeter values. (C) The mRNA expression levels of TWEAK and Fn14 were determined in fresh tissues. (D) The mRNA levels of regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein 1 (MCP-1), and interferon gamma-induced protein-10 (IP-10) were also determined in these tissues. (E) Confocal microscopy was performed for Ro52 and Fn14 expression in lesional skin. Number of normal samples = 10, number of non-lesional samples = 15, number of lesional samples = 15. Data points and error bars represent mean ± SEM. Representative images are shown. Bar = 50 µm. *p < 0.05, **p < 0.01.