Additional file 1 of A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells
Creators: name: Hanel, Walter
name: Lata, Pushpa
name: Youssef, Youssef
name: Tran, Ha
name: Tsyba, Liudmyla
name: Sehgal, Lalit
name: Blaser, Bradley W.
name: Huszar, Dennis
name: Helmig-Mason, JoBeth
name: Zhang, Liwen
name: Schrock, Morgan S.
name: Summers, Matthew K.
name: Chan, Wing Keung
name: Prouty, Alexander
name: Mundy-Bosse, Bethany L.
name: Chen-Kiang, Selina
name: Danilov, Alexey V.
name: Maddocks, Kami
name: Baiocchi, Robert A.
name: Alinari, Lapo
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 1: Fig. S1. (Left) B-cell were isolated from the peripheral blood from healthy donors and cultured in the absence (left) or presence (right) of cytokines and a CD40L expressing fibroblasts as previously described((1)). Flow cytometry was performed for the indicated markers. (Top right) Resting and activated B cells were fixed and stained with PI for cell cycle analysis. (Bottom right) Lysates were collected from resting and activated B-cells and blotted for p-Btk, total Btk, c-myc, and GAPDH. Fig. S2. (top) UMAP plots from 4 different patients with leukemic MCL showing lineage specific clustering of cells. (bottom) Dot plots verifying lineage specific marker enrichment among the individual clusters. Fig. S3. UMAP plots compiled from 4 different patients with leukemic MCL (left) or a reference PMBC B-cell data set (right) showing their distinct transcriptional states with an overall small amount of normal B-cells in leukemic MCL samples. Fig. S4. Jeko cells were retrovirally transduced with lentiviruses encoding either a non-targeting sh or an sh directed against SAE1. Cells were GFP sorted and the total viable cells in each was enumerated 3 days after sorting (n=3 biological replicates). Fig. S5. B cells and MCL cell lines were treated with TAK-981 (100 nM, 24 hours). Lysates were prepared and blotted for an antibody recognizing the SUMO-TAK-981 adduct (above) and GAPDH (below). Fig. S6. Representative cell cycle profiles of primary MCL patients samples taken at the time of beginning TAK-981 treatment in Fig.3B. Fig. S7. Jeko cells were synchronized with palbociclib (500 nM) and treated with either DMSO or TAK-981 (100nM) and lysates. Cell were fixed at the indicated time points and cell cycle distribution was determined of PI stained cells. Fig. S8. Z-138 cells were synchronized with palbociclib (500 nM) and treated with either DMSO or TAK-981 (100nM) and lysates. Cell were fixed at the indicated time points and cell cycle distribution was determined of PI stained cells. Fig. S9. (Top) Z-138 cells were synchronized with Palbociclib (500 nM, 24 hours) followed by drug washout and treatment with nocodazole (50 ng/mL) either in the presence of DMSO or TAK-981 (100 nM) for 24 hours. Both drugs were then washed out and cells were collected every hour for cell cycle analysis and lysates were prepared for protein for SUMOylation levels. (Bottom) Z-138 cells were synchronized with Palbociclib (500 nM, 24 hours) followed by drug washout and treatment with nocodazole (50 ng/mL) for 24 hours. Cells were then treated with either DMSO or TAK-981 for 3 hours. Cells were then washed from drugs and treated with either DMSO or TAK-981 and collected every hour for cell cycle analysis and lysates were prepared for protein for SUMOylation levels. Fig. S10. Results of DAVID functional annotation analysis performed on proteins enriched for sumoylation for SUMO1 (top) and SUMO2/3 (bottom). The percentage of SUMOylated proteins within individual functional annotations are shown along with the false discovery rate (FDR). Fig. S11. (Top). Jeko cells were synchronized with Palbociclib (500 nM, 24 hours) followed by drug washout and treatment with nocodazole (50 ng/mL) either in the presence of DMSO or TAK-981 (100 nM) for 24 hours. Both drugs were then washed out and cells were fixed and stained for CENP-A. Representative confocal images of cells or chromosomes are shown. (Bottom) Enumeration of CENP-A foci from DMSO or TAK-981 treated cells was performed using cellSens software. Results are representative of two independent experiments.