Extended Data Fig. 8: Control of MYC multimerization by ubiquitylation and SUMOylation.
From: MYC multimers shield stalled replication forks from RNA polymerase
a. Confocal immunofluorescence of indicated proteins in U2OS cells expressing WT MYC or K-less MYC. Treated as in Fig. 2d. Scale bar: 20 µm (n = 3). b. MYC-MYC PLA in U2OSMYC-AID cells reconstituted with WT MYC or K-less MYC as indicated. Endogenous MYC depleted by addition of indoleacetic acid (500 µM, 6 h). PLA (red) is overlaid with nuclear staining (blue) and compared with MYC staining (green). Scale bar: 5 µm (n = 3). c. Immunoblot of endogenous K6-linked ubiquitin immunoprecipitations from U2OS cell extracts. Immunoprecipitated K6-linked ubiquitin was visualized using a α-GFP antibody detecting the K6 affimer. Actin was used as a loading control, *IgG heavy chain (n = 1). For gel source data, see Supplementary Fig 1. d. Quantification of PLAs between MYC and indicated proteins in DOX (1 µg/ml, 24 h) and PlaB (1 µM, 4 h) treated U2OSMYC-Tet-On cells. Signals are corrected for corresponding single antibody control and presented as fold change to untreated. PLA data were analyzed for the following total number of cells: PLA between MYC and K48 (DMSO: n = 750, PlaB: n = 754), K6 (DMSO: n = 675, PlaB: n = 747), USP28 (DMSO: n = 657, PlaB: n = 554), USP36 (DMSO: n = 799, PlaB: n = 644), HUWE1 (DMSO: n = 343, PlaB: n = 477) analyzed over three independent experiments and are expressed as mean ± S.E.M. Two sided Mann-Whitney U test was used for comparison to untreated cells with following p-values: K48 = 3.3e-11, K6 = 1.8e-3, USP28 = 5.6e-3, USP36 = 1.3e-10, HUWE1 = 5.5e-3. e. Representative images of confocal immunofluorescence of MYC with USP28-MYC and USP36-MYC PLAs within the same cells at indicated treatments. Scale bar: 5 µm. f. Dose response curve of MYC multimer formation in response to BI8626 treatment at indicated concentrations using a four-parameter Weibull function (n = 3). g. Confocal immunofluorescence of MYC and SUMO2/3 in U2OSMYC-Tet-On cells treated as indicated (DOX: 1 µg/ml, 24 h; MG-132: 20 µM, 4 h; ML-792: 1 µM, 24 h). Scale bar: 5 µm (n = 3). In above panels, unless indicated otherwise, n indicates the number of independent biological replicates.
