Fig 1: PRMT1 does not change LDC expression and composition but modulates LDC methylation. (A) Workflow of the experimental setup designed for proteomics analysis of LDC expression and composition, upon PRMT1 modulation. Co-IP of the LDC using nuclear extracts from a 1:1 mix of SILAC-labelled HeLa cells, depleted of- or overexpressing PRMT1 (KD, sh-1 and PRMT1 OE), or infected with the EV (control). Each immuno-enrichment of the complex with individual baits was carried out in three independent biological replicates (n = 3), with SILAC channels swapping (Forward and Reverse experiments). (B) LDC expression analysis: scatterplot of all proteins identified and quantified in the SILAC nuclear inputs of HeLa cells, depleted or not of PRMT1. The scatterplot displays the log2 SILAC distribution of the quantified proteins in the Forward (Fwd) and Reverse (Rev) experiments (H/L ratio for the Fwd and L/H ratio for the Rev experiment). The dot corresponding to PRMT1 is highlighted in blue; LDC subunits are displayed in orange and all remaining proteins are in grey. Dashed lines indicate the µ ± 2? cut-off, calculated from the whole peptide SILAC ratio distribution in both Fwd and Rev experiments, that separate significant outliers form the unchanging population. (C) LDC protein composition analysis: scatterplots of all proteins identified and quantified in the DGCR8 co-IPs, analyzed as described in B; the dot corresponding to DGCR8 is displayed in blue; all LDC proteins are indicated in orange, the remaining proteins are in grey. Dashed lines represent the µ±2? cut-off calculated on the protein ratio distribution, and define significantly changing from unchanging proteins. (D) Heat map display of the unsupervised clustering analysis of methylated peptides identified and quantified in the SILAC/co-IP experiments upon PRMT1 KD and PRMT1 OE. Only peptides identified in at least two co-IP experiments of either the PRMT1 KD or PRMT1 OE were included in the analysis. Data are displayed as the average from three independent IPs for each bait and expressed as log2 ratio of PRMT1 KD/OE cells, compared to control cells. The heat map shows an unsupervised clustering of the data, generated using Pearson correlation and data average analysis. The type of methylation identified for each methyl-peptides is displayed (me = mono-methylation; di = di-methylation; tr = tri-methylation), as well as the respective methyl-sites. The 2 main clusters identified are indicated with differential color code (cluster A in red and cluster B in blue). * = Methylated peptides significantly regulated in the SILAC experiments upon a µ ± 1? cut-off calculated from the unmodified peptide distribution of each IP experiment. (E) Motif enrichment analysis of all the SILAC-quantified methyl-peptides using ScanX software (115). The following parameters were imposed: R central character; width = 7; occurrences = 10; significance = 0.000001 (right panel). The same analysis was performed also on the methyl-peptides identified in the cluster A and cluster B, separately (right panel). (F) LDC complex representation with Cytoscape of the proteins identified in the SILAC/co-IP experiments: proteins found methylated are displayed in green. Methylated proteins whose methylation state is significantly changing upon PRMT1 modulation are highlighted with orange circles. Non-methylated proteins are displayed in gray. (G) Immuno-enrichment of ILF3 and TAF15 from HeLa cells treated with DMSO, the MS023 inhibitor and the MS094 control compound, used at 10 µM concentration for 16 h. WB profiling using anti-ADMA, anti-ILF3 and anti-TAF15 to assess the level of di-methylation and unmodified ILF3 and TAF15 protein levels, respectively.