Fig 2: RNF111-dependent Proteome.A, schematic representation of the proteome experiment with a summary table of the RNF111-RING-KO/U2OS protein ratio quantification results. B, volcano plot representation of the differential analysis with the log2 fold change RNF111-RING-KO/U2OS versus the negative log10p-value. Each dot represents a protein. Green and red lines represent the cut-off applied (respectively fold increase or decrease =2 and p-value = 0.05). Volcano Plot is zoomed in the fold increase = 2 and p-value = 0.05 areas. C, Western blot analysis of the proteome candidates. U2OS cells and RNF111-RING-KO clones #1 and #2 were treated or not with TGF-ß for 1 h. The whole cell extracts were analyzed by Western blotting using antibodies against RNF111, SKIL, SKI, GDF15 (growth differentiation factor 15), KYNU, FABP3 (fatty acid binding protein 3), and GAPDH. D, qPCR analysis of the proteome candidates. U2OS cells and RNF111-RING-KO clones #1 and #2 were treated or not with TGF-ß for 1 h. The levels of mRNA for SKIL, GDF15, KYNU, and FABP3 were analyzed by qPCR and normalized to GAPDH using the 2-??Ct methods. The data represent mean ± SD for at least three independent experiments. KYNU, Kynureninase; RNF111, ring finger protein 111; TGF-ß, transforming growth factor-ß.

Fig 3: Comparison of diGly and pan UB ubiquitylomes.A, the heatmap represents the ratios for the significant RNF111 substrate candidates in the pan UB ubiquitylome compared with the diGly ubiquitylome ratios. The ratios are annotated as fold increase or decrease, indicated by the arrows. Asterisks indicate their p-value: *p = 0.05, **p = 0.01, and ***p = 0.001. Note that we compared whole protein quantification in the pan UB case against ubiquitylation sites quantification for diGly (protein values may then be repeated when corresponding to multiple sites). B, Venn diagram comparison shows four common hits, including SKIL, between the pan UB and diGly ubiquitylomes significant RNF111 substrate candidates. diGly, diglycine; UB, ubiquitin.

Fig 4: Identification of RNF111-dependent substrates by diGly ubiquitylome quantitative comparison.A, schematic representation of the experimental design for RNF111-dependent ubiquitylome quantification with the diGly approach using immunoprecipitation of ubiquitylated trypsic remnant peptides with the K-e-GG antibody. The table summarizes the results for the ubiquitylation sites quantification of the RNF111–RING–KO/U2OS ratio. B, volcano plot representation of the differential analysis with the log2 fold change RNF111-RING-KO/U2OS versus the negative log10p-value. Each dot represents a ubiquitylation site. Green and red lines represent the cut-off applied (respectively fold increase or decrease =2 and p-value = 0.05). Volcano Plot is zoomed in the fold decrease = 2 and p-value = 0.05 area. C, schematic representation of the SKIL protein with its domains DHD, SAND, and CC (coild-coild) and the localization of lysine 343. Lysines 342 and 343 that were subsequently mutated are indicated in red. D, HEK-293 cells were transfected with Flag-RNF111-WT or Flag-RNF111-C933A catalytic inactive mutant along with HA-SKIL-WT, HA-SKIL-342/43-KR mutant, or empty vector. After 1 h Activin/TGF-ß treatment, the whole cell extracts were immunoprecipitated with the pan UB nanobody and analyzed by Western blotting (pan UB, right panel) along with the corresponding whole cell extract (input, left panel) using HA and Flag antibodies. diGly, diglycine; RNF111, ring finger protein 111; UB, ubiquitin.

Fig 5: CRISPR engineered U2OS RNF111-RING-KO cell lines.A, upper diagram, design of the reverse and forward sgRNA (sgRNA-rev and sgRNA-fw, in red) used to target exon 5 of RNF111 gene in U2OS cells. Red arrows indicate the breaking sites. Amino acids of the corresponding codons are annotated below; nonsense mutation on serine 432 observed in NCI-H460 cell line is indicated (S432*). Lower diagram, schematic representation of WT RNF111 in parental U2OS cell line compared with RNF111 truncation in U2OS CRISPR engineered RNF111-RING-KO clones #1 and #2 and NCI-H460 cell line. B, U2OS cells, NCI-H460 cells, and RNF111-RING-KO clones #1 and #2 were treated or not with TGF-ß for 1 h. The whole cell extracts were analyzed by Western blotting using antibodies against RNF111, SMAD2/3, P-SMAD2, SKIL, SKI, and GAPDH. C, U2OS cells, NCI-H460 cells, and RNF111-RING-KO clones #1 and #2 were cotransfected with the CAGA12-Luc and pRL-TK reporters and were treated or not with TGF-ß for 8 h. The data represent means ± SD of luciferase activities normalized to Renilla in triplicate experiments. RNF111, ring finger protein 111; TGF-ß, transforming growth factor-ß.