Fig 2: SMURF2 Ser384 phosphorylation affects the SMURF2-induced RNF20 ubiquitination.A, the RNF20 protein levels were similar among Smurf2−/− MEFs stably expressing control vector pBabe, SMURF2 (WT), or SA upon cycloheximide treatment. B, upon both cycloheximide and etoposide treatment, the RNF20 protein level was decreased in Smurf2−/− cells expressing SMURF2 (WT), compared with the RNF20 protein level in the cells expressing control vector or SMURF2 (SA). C, ATM inhibitor restored the RNF20 protein level in Smurf2−/− cells expressing SMURF2 (WT) in the presence of cycloheximide and etoposide treatment. Quantitation of RNF20 protein levels from three independent experiments is shown in the right panels. D, the interaction between RNF20 and SMURF2 is regulated by ATM and etoposide (Etop) treatment. Control (shLuc) or ATM knockdown (shATM) USOS cells were treated with DMSO or etoposide and then subjected to SMURF2 IP. The presence of RNF20 was examined by Western blotting. WCL, whole cell lysate. E, RNF20 preferentially interacts with SMURF2 (WT). Smurf2−/− MEFs, which were stably expressing FLAG–SMURF2 (WT or SA) or pBabe vector, were subjected to FLAG IP and Western blotting analysis. F, SMURF2 (WT) but not SA is required for the polyubiquitination of RNF20. HA–ubiquitin was transfected to Smurf2−/− MEFs expressing SMURF2 (WT) or SA mutant, and the cells were treated with MG132 with or without etoposide. After RNF20 IP, the ubiquitination signal was visualized by Western blotting.

Fig 3: Effect of IL2-Smurf2 and *716 on treated cells: (A) L1210 cells were treated with 0.05 µM MG132 or were untreated for 24 h and then 6 µg/mL IL2-Smurf2, 8 µg/mL *716 or PBS was added and the cells incubated for a further 24 h. Western blot analysis was performed with anti-FK2 and anti-β actin antibodies; (B) Quantitation of (A); (C) HUT102 cells were treated with 6 µg/mL IL2-Smurf2, 8 µg/mL *716 or PBS and incubated for 24 h. Western blot analysis was performed with anti-K48 and anti-β actin antibodies; (D) Quantitation of (C); (E) L1210 cells were treated with 6 µg/mL IL2-Smurf2, 8 µg/mL *716 or PBS and incubated for 24 h. Western blot analysis was performed with anti-K48 and anti-β actin antibodies; (F) Quantitation of (E); (G) L1210 cells were treated with 6 µg/mL IL2-Smurf2, 8 µg/mL *716 or PBS and incubated for 24 h. Western blot analysis was performed with anti-RNF20 and anti-β actin antibodies; (H). Quantitative of 3 repetitions of G. * p < 0.03, ** p < 0.01. The uncropped Western Blot images can be found in Figure S3.

Fig 4: The core circadian component BMAL1 regulated histone H2B monoubiquitination levels(A) RNA-seq heatmap comparing the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 7th day of osteogenic differentiation. (B) GO analysis of the RNA-seq data between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups. Bar graph showing the p values of the enriched terms. (C) GSEA of the RNA-seq data between the Sh-NC and Sh-BMAL1, Sh-NC, and Sh-CLOCK groups. (D) H2Bub1 and H2Aub1 levels in the MSCs infected with Sh-NC, Sh-BMAL1, or Sh-CLOCK lentiviruses on the 10th day of osteogenic differentiation. H2B and H2A served as the internal controls. Bar graphs showing the relative levels. Data are presented as mean ± SD; n = 3; ∗p < 0.05. (E) log2 FC and −log10(q value) of differential RNF20/40 expression between the Sh-NC and Sh-BMAL1 groups and the Sh-NC and Sh-CLOCK groups as obtained from the RNA-seq data. (F) Circos plot showing the terms with enriched genes and log2 FC and −log10(q value). TTK, the regulator of histone H2B monoubiquitination, is highlighted (G).

Fig 5: Let-7b-5p inhibitor reverses the exosome-induced decrease of RNF20 expression in C2C12 myotube cells. (A–D) Western blotting results of RNF20 expression in C2C12 myotube cells co-cultured with KPC exosomes (left) or Pan02 exosomes (right) after treatment with let-7b-5p inhibitor and quantification of the protein bands, and β-actin was used as a control; (E–F) qRT-PCR detection of RNF20 mRNA expression in C2C12 myotube cells treated as in A and B. *P < 0.05, **P < 0.01.