Extended Data Fig. 5: NuMA-L promotes SSBR and transcription recovery functions.
From: A mechanism for oxidative damage repair at gene regulatory elements
(a) A schematic of human NuMA isoforms. (b) GFP- immunoprecipitates from GFP-NuMA-L and NuMA-S-transfected HEK-293 cells, treated with H2O2 and subjected to mass spectrometric analysis. Annotated tandem mass spectra of NuMA-L (top) and NuMA-S (bottom) specific peptides were identified in the immunoprecipitates. Precursor mass deviation was 2.07 ppm for the peptide LTAQVEQLEVFQR and 0.66 ppm for the peptide LTAQVEELSK. (c) Quantitative enrichment of interacting proteins between NuMA-L and NuMA-S from (b). NuMA-interacting proteins were identified from statistical analysis using Student t-test. Solid lines indicate enriched interacting proteins after filtering for FDR of 0.05 and an artificial within groups variance, S0 = 1. Annotated blue and red dots indicate NuMA-S (STR), NuMA-L (LTR) and additional proteins of interest. Non-annotated pale red dots represent proteins significantly enriched in each NuMA pull down compared to GFP controls. Green dots are proteins enriched in both NuMA-S and NuMA-L pull-downs and red dots are proteins enriched only in NuMA-L pull-downs. (d) Immunoblotting of GFP- immunoprecipitates from GFP-empty, GFP-NuMA-L and GFP-NuMA-S-transfected HEK-293 cells, treated with H2O2, n = 3 biological replicates. (e) Immunoblotting of transfected MRC5 cells., n = 5 biological replicates. (f) MRC5 cells were transfected as in (e), treated with H2O2 and recovered in media. A representative plot showing spread of comet tail moments from 250 nuclei (two-sided, unpaired, t-test). (g) Bars represent percentage DNA strand breaks remaining. Error bars (±s.e.m,), n = 5 biological replicates (two-sided, unpaired t-test). (h) Clonogenic survival of MRC5 cells transfected as in (e) and presented on a semi-log scale, n = 3 biological replicates (two-sided, unpaired t-test). (i) Immunoblotting of transfected MRC5 cells. (j) MRC5 cells were transfected as in (i) and nascent transcripts labelled with EU. Scatter plots show fold change of average fluorescence. Error bars (±s.e.m.), n = 3 biological replicates (two-sided, unpaired t-test).
