Fig 1: Fosl1 interacts with JunB and promotes ccnt1 expression during heart regeneration in X. tropicalis.a Heat map of log2(FC) values showing genes differentially expressed in the hearts at 0.5, 1, 7, 14, 30, and 60 dpr compared with sham. b The qPCR of some selected regulators for cell cycle progression in sham and 0.5 dpr hearts (n = 4 each). c GSEA analysis of hearts at 0.5 dpr showing high correlations with Fosl1 (left) and JunB (right) pathways. d Expression of Flag-tagged xFosl1 (left) and HA-tagged xJunB (right) in X. tropicalis embryos was confirmed by western blotting. e CoIP assay showing the interaction between xFosl1-Flag and xJunB-HA in X. tropicalis embryos. f CoIP analysis showing the increased interaction between Fosl1 and JunB in regenerating neonatal mouse heart compared with the quiescent heart. g qPCR validation of cell cycle regulators with significant upregulation in RNA-seq analysis (n = 4 each). h Promoters of the cell cycle regulators with more than twofold upregulation were subjected to luciferase activity assay, using reporter plasmids and mutants (n = 5 each). i GSEA analysis of the hearts at 0.5 dpr showing high correlations with Ccnt1 pathway. j Representative image of ChIP assay confirmed the in vivo interaction of Fosl1 with promoters of ccnt1 in X. tropicalis embryos. k ChIP-qPCR analysis showing the increased interaction between Fosl1 protein and Ccnt1 promoter region in regenerating neonatal mouse heart compared with quiescent heart. l, m qPCR validation of Fosl1 (l) and JunB (m) in sham and resected hearts of neonatal mice (n = 4 each). All data are presented as mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 versus control (Student’s t test).
Fig 2: JunB becomes FAT10ylated at a SUMOylation consensus site. a JunB-FAT10ylation under overexpressing conditions in HEK293 cells. Cells were transiently transfected with expression plasmids for the proteins indicated and an immunoprecipitation (IP) against HA-FAT10 combined with western blot (IB) analysis was performed. 10 µM proteasome inhibitor MG132 was added for a total of 6 h before harvesting, where indicated. ß-actin was used as loading control. Shown is one experiment out of three experiments with similar outcomes. b Conjugation of FAT10 to JunB under endogenous conditions in human PBLs. Endogenous FAT10 expression was induced by IFN-?/TNF-a treatment for 48 h and JunB-FAT10 conjugate formation was analyzed in a combined immunoprecipitation/western blot analysis. Shown is one experiment out of three experiments with similar outcomes. The asterisk marks an unspecific background band. Mouse IgG1 was used as isotype control for the IP. Bar graph represents relative expression levels of FAT10 mRNA, normalized to the housekeeping gene GAPDH as measured by real-time PCR. Ct-levels of untreated cells were 22.4, and 21.7 for IFN-?/TNF-a treated cells. c The degradation rate of the JunB-HA-FLAG-FAT10 conjugate in transiently transfected HEK293 cells was monitored by cycloheximide (CHX) chase experiments. Cells were treated for 2.5 or 5 h with 50 µg/ml CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for a total of 6 h. Immunoprecipitation and western blot analysis was performed as described in a. d Densitometric analysis of ECL signals of CHX chase experiments as shown in c. ECL levels were normalized to the ECL signals of ß-actin and the level of 0 h CHX was set to unity. Values are shown for five independent experiments with similar outcomes as means ± s.e.m. e FAT10 becomes conjugated to a lysine within one of the three reported SUMOylation consensus sites in JunB. Western blot analysis of JunB-HA-FLAG-FAT10 conjugates in transiently transfected HEK293 cells upon expression of wild-type JunB or of JunB mutants, in which single lysines within three different SUMOylation consensus sites or all three (JunB-K3R-HA) were mutated to arginines. Immunoprecipitation and western blot analysis was performed as described in a. Shown is one experiment out of three experiments with similar outcomes. Source data are provided as a Source Data file
Fig 3: Expression of proteins in C2C12 myotubes. Representative immunoblots are shown on the left side, and the quantification of MyHC-fast (A), MyHC-slow (B), myogenin (C), MyoD (D), JunB (E), myostatin (F), MAFbx (G), and MuRF-1 (H) was determined after normalizing to GAPDH. * p < 0.05 vs. con.
Fig 4: Initial injury is characterized by cell stress, transcriptional regulation and cell adhesion. (a) Gene ontology enrichment identified inflammatory response and cell adhesion as significantly upregulated processes, among others, 2 hours after injury. (b) The top 50 differentially expressed genes (identified by lowest p-value) organized by fold change. (c) Heat map showing differences in normalized counts of the top 50 differentially expressed genes between no injury (n = 5) and 2 hours (n = 5). (d) Protein-protein interaction network of top 50 differentially expressed genes via STRING analysis identified clusters specific to cell stress and cell adhesion. (e) JunD expression 2 hours following injury (green) as revealed by immunohistochemistry. VE-cadherin and DAPI denote endothelial junctions (red) and nuclei (blue), respectively. Scale bar: 60 µm (f) Immunohistochemistry for JunB expression 2 hours following injury (green). VE-cadherin and DAPI reveal endothelial junctions (red) and nuclei (blue), respectively. Scale bar: 60 µm.
Fig 5: JunB SUMOylation is diminished in the presence of FAT10. a Western blot showing in vitro FAT10ylation and SUMOylation of JunB. SUMO-1 or -2 conjugation to JunB is completely inhibited in the presence of FAT10 or FAT10-AV. Recombinant proteins were incubated at 30 °C for 50 minutes in in vitro buffer, supplemented with ATP. Reactions were stopped by addition of gel sample buffer, supplemented with 10% 2-mercaptoethanol (2-ME) and boiled. Shown is one experiment out of three experiments with similar outcomes. The recombinant protein amounts used can be found in the methods section. Asterisks mark unspecific background signals in lanes 1, 4, 5 and 9. b In cellulo SUMOylation of JunB is diminished in the presence of FAT10. Stably FLAG-FAT10 expressing HEK293T cells were transiently transfected with HA-tagged JunB or JunB-K3R and an anti-HA immunoprecipitation combined with western blot analysis was performed. Shown is one experiment out of three experiments with similar outcomes. Source data are provided as a Source Data file
Supplier Page from Abcam for Anti-JunB antibody [EPR6518]