Fig. 6.

p38-dependent phosphorylation of NELFE promotes its dissociation from chromatin. a Chromatin-associated proteins were extracted from untreated, UV-light-treated, and p38i, UV-light-treated U2OS cells, and analyzed by SILAC-based quantitative mass spectrometry. The bar plot shows the GO-BP (biological process) terms associated with proteins specifically enriched on or removed from chromatin after UV light (40 J/m², 1 h recovery). The significance of the enrichment of a specific term was determined using Fisher’s exact test. P-values were corrected for multiple hypotheses testing using the Benjamini and Hochberg FDR. b The bar plot shows selected proteins associated with DNA repair and cell cycle that are significantly recruited or removed from chromatin after UV light. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t-test was used to assess the significance. c The NELF complex subunits are removed from chromatin in a UV light and p38-dependent manner. The bar plot shows selected proteins whose removal from chromatin after UV light is dependent on p38. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t-test was used to assess the significance. d NELFE dissociates from chromatin after UV light. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies (left). The levels of NELFE on chromatin were quantified from three replicate experiments and normalized to MCM7 levels (right). The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t-test was used to assess the significance (***p-value < 0.001, **p-value < 0.01). e Dynamics of NELFE removal from chromatin. Chromatin protein fractions from differentially treated U2OS cells were resolved by SDS-PAGE and subjected to western blotting with the indicated antibodies. The error bars show the mean and SD of SILAC ratios quantified from three replicate experiments. Two-sided Student’s t-test was used to assess the significance (**p-value < 0.01). f Knockdown of NELFE reduced the ability of U2OS cells to form colonies after UV light. The error bars show the mean and SD of results obtained in three replicate experiments performed in three technical replicates. Two-sided Student’s t-test was used to assess the significance (***p-value < 0.001, **p-value < 0.01)