Extended Data Fig. 9: Role of HUWE1 in MYC multimerization.
From: MYC multimers shield stalled replication forks from RNA polymerase
a. Representative MS spectrum visualizing MYC ubiquitylation at K148 and K389 in diGLY experiments performed upon treatment with PlaB (2 h, 1 µM), CBL0137 (2 h, 5 µM) or HU (4 h, 5 mM) (n = 3). b. Immunofluorescence pictures of U2OS cells expressing MYCK-R148KR389K. Scale bar: 5 µm (n = 3). c. Fiber assay assessing unperturbed DNA replication (T1) in U2OSMYC-AID expressing either WT, K-less or no MYC. Mean of n = 118 fibers (MYC OFF), n = 127 (WT MYC) and n = 110 (K-less MYC) from one representative of three experiments with similar results is shown ± S.E.M. p-value: Mann-Whitney U test, two-sided. d. BLISS assays in U20SMYC-AID cells reconstituted with MYCK-R148KR389K. MYC OFF samples are the same as shown in Fig. 4f (n = 2). e. Box plot showing quantification of spike normalized reads from EXOSC10 CUT&RUN in U2OSMYC-Tet-On cells on 845 genes, which show increased antisense transcription in absence of MYC. Cells were treated as indicated (MG-132: 20 µM, 4 h; BI8626: 10 µM, 4 h). Box plots are defined as in Fig. 3g. Mann Whitney U test, one-sided: ns, not significant; ****p-value < 0.0001. f. Fiber assay assessing fork degradation (T2) upon treatment with BI8626 (20 µM, 4 h) and Mirin (50 µM, 4 h) in KPC and U2OS cells. Mean of n = 177 (CTL), n = 126 (BI8626), n = 148 (CTL+Mirin) and n = 151 (BI6826+Mirin) fibers from one representative of three experiments with similar results is shown ± S.E.M. for KPC cells (left). Mean of n = 136 (CLT) and n = 72 (BI8626) fibers ± S.E.M. in U2OS cells shown from one representative of three experiments with similar results (right). p-value: Mann-Whitney U test, two sided. g. Fiber assay assessing fork restart (T3; see Extended Data Fig. 7g) upon treatment with BI8626 (20 µM, 4 h) in KPC (left) and U2OS (right) cells. Mean ± S.D. of three independent biological replicates is shown with each replicate quantifying more than 500 fibers per condition (CTL: 505, 506, 501; BI8626: 502, 507, 523) in KPC and more than 400 fibers (CTL: 408, 407, 432; BI8626: 426, 456, 495) in U2OS cells. p-value: unpaired t-test, two sided. In above panels, unless indicated otherwise, n indicates the number of independent biological replicates.
