Fig 2: Overexpression of MKK7AC to promote the phosphorylation of JNK2 inhibited the metastasis of 4T1 cells(A) Western blot results of MKK7, p-JNK, JNK2 and Fra1 in 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells. (B) Transwell assay results of 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells (n = 3). (C) Wound healing assay results of 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells (n = 8). (D) Fra1-reporter luciferase activity in 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells, n = 3. (E) Immunofluorescent staining of E-cad (red), N-cad (green) in 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 cells, the nuclei were counterstained with DAPI (blue). Scale bar: 50 µm.

Fig 3: JNK2 regulated the transcript of Fra1 through c-Jun and Jun-B, which bound to the promoter of Fra1(A) Western blot of c-Jun, Jun-B, p-c-Jun (Ser63), p-c-Jun (Ser73), and JunD in 4T1 and EO771 cells with or without JNK2 silencing (A). (B-D) Western blot of c-Jun and Jun-B in the cells below: EO771 and ZR-75-1 cells treated with SP600125 and DMSO control for 24h (B); EO771-sc, EO771-shMEKK1-1 and EO771-shMEKK1-1-shJNK2-1 (C) ; 4T1-sc, 4T1-MKK7AC and 4T1-MKK7AC-shJNK2-1 (D). (E) The mRNA expression changes of c-Jun and Junb were examined in EO771-sc and EO771-shJNK2 cells by real-time quantitative RT-PCR (upper panel, n = 3). The ubiquitination of c-Jun and Jun-B were examined through co-IP in EO771-sc and EO771-shJNK2-1 cells (lower panel). (F) Western blot of c-Jun, Jun-B and Fra1 in 4T1 with c-Jun or Junb down-regulation and their control cells. (G) The visual representation of predicted c-Jun-and-Jun-B targets on Fra1 promoter region (P1: -17.732 kb, P2: -17.648 kb, P3: 199 bp, P4: 1157 bp and P5: 2597 bp). Five pairs of primer were designed for quantitative-PCR (Q-PCR) to detect the binding activity. (H) ChIP and Q-PCR analysis were performed in 4T1-wild type (wt) with anti-c-Jun and anti-Jun-B antibody. Binding enrichment was normalized to the input DNA (n = 3). (I) Fragments of Fra1 promoter with or without P3 position were constructed and cloned into the PGL3 basic vector and were named as F1-Luc and F2-Luc separately. (J) Statistical results for the promoter activity of F1-Luc and F2-Luc in 4T1-wt, EO771-sc, EO771-shMEKK1-1, n = 3 for 4T1, n = 6 for EO771.

Fig 4: USP21 regulates degradation of Fra-1 protein. (A–C) To determine Fra-1 ubiquitination, Fra-1 plasmid was co-transfected with HA-Ub and SRT-USP21 wild type (WT) or SRT-USP21 C221A in 293T cells. The total cell lysates were immunoblotted with the indicated antibodies (A) and followed by immunoprecipitation (IP) with anti-Fra-1 antibody (B). Ubiquitinated Fra-1 level was determined by immunoblotting with anti-ubiquitin or anti-HA antibody before and after IP. Expression of ß-actin was used as a loading control. (C) After transfection with siUSP21 for 24 h, 293T cells were co-transfected with plasmids encoding HA-Ub, Fra-1, and SRT-USP21 WT and treated with MG-132 for 6 h before cell harvesting. The cell lysates were immunoprecipitated with anti-Fra-1 antibody and subsequently immunoblotted with anti-HA antibody. Total cell lysates were immunoblotted with indicated antibodies, and expression of ß-actin was used as a loading control. (D) HCT116 cells were transfected with empty vector (control) and SRT-USP21 for 24 h, after which cycloheximide (CHX) was applied for the indicated times. The expression levels of Fra-1, USP21, and GAPDH were measured by immunoblotting, and quantification of band intensities from triplicate determinations is shown at the bottom. Significance was determined using two-way ANOVA test between two groups (*** p < 0.001, ** p < 0.01, * p < 0.05).

Fig 5: USP21 regulates Fra-1 activities. (A,B) HCT116 and HT-29 cells were transfected with scrambled siRNA (control) or siUSP21 for 24 h and then co-transfected with plasmids encoding Fra-1 or SRT-USP21 as indicated. After introducing AP-1 (A) or MMP-1 reporter plasmid (B) for an additional 24 h, luciferase transcriptional responses were measured in each cell line. Data were statistically analyzed by one-way ANOVA (n = 3). (C) After transfection with indicated plasmids into HCT116 and HT-29 cells, MMP-1 mRNA expression was quantified by real time-PCR with normalization to GAPDH. Values are fold-increases relative to the control, and error bars represent mean ± SD. Data were statistically analyzed by one-way ANOVA (n = 4). (D) HCT116 and HT-29 cells were transfected with scrambled siRNA (control) or siUSP21 for 24 h and subsequently co-transfected with plasmids encoding Fra-1 or SRT-USP21. Total cell lysates were analyzed by immunoblot analysis with the indicated antibodies. Quantification of band intensities was also shown by graph (n = 3–4). ** p < 0.01 vs. control, ## p < 0.01 vs. Fra-1, and # p < 0.05 vs. Fra-1.