Journal: Experimental Hematology & Oncology

Article Title: A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells

doi: 10.1186/s40164-022-00293-y

Figure Lengend Snippet: Loss of sumoylation results in mitotic dysregulation with accumulation of polyploid cells A (left). Volcano plot of genes with a statistically significant association with SAE1 and SAE2 (FDR < 0.01). (right) DAVID analysis was performed on the statistically significant gene list. The top gene groups and enrichment scores are shown. B (right). Normal and activated B-cells and MCL cell lines were treated with DMSO, 50 nM or 100 nM of TAK-981 and cells were collected daily. Cells were fixed, stained with PI and cell cycle analysis was performed. Representative DNA profiles are shown for the indicated cell lines and primary samples treated with 100 nM of TAK-981. Quantification of 2n cells relative to DMSO control (top, right) at 24 h and percentage polyploid cells (bottom, right) after 48 h of TAK-981 treatment at the indicated concentrations (n = 3 independent experiments per cell line). C Primary MCL samples were treated and analyzed as in B (n = 3 independent experiments per sample). D (Left) The indicated cell lines were treated with palbociclib (200 nM) for 24 h and then released into DMSO, palbociclib, TAK-981 or palbociclib + TAK-981. (right top) Percentage viable cells was quantified after 72 h from the time of release from the initial palbociclib treatment (n = 3 independent experiments for each cell line, * p < 0.05). (right bottom) Western blot confirming equivalent losses of sumoylated proteins with TAK-981 in the presence or absence of TAK-981

Article Snippet: For SAE1 knockdown, after lentiviral transduction from viruses obtained from transfection of SAE1 human shRNA plasmids (Origene, Cat#TL315567), cells were GFP sorted 1 day after transfection and downstream assays were carried out after three days from sorting.

Techniques: Staining, Cell Cycle Assay, Control, Western Blot

Journal: Experimental Hematology & Oncology

Article Title: A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells

doi: 10.1186/s40164-022-00293-y

Figure Lengend Snippet: Inhibition of SUMO conjugation during S phase is required for proper mitotic division in MCL cells A Jeko (left) or Z-138 (right) cells were synchronized with palbociclib (500 nM) and treated with either DMSO or TAK-981 (100 nM) and lysates were prepared at the indicated time points after washout. The corresponding phase of the cell cycle based on the DNA profiles obtained from fixed, PI stained cells from each of the time points is shown. Lysates were blotted for SUMO1, SUMO2/3, SAE1, SAE2, UBC9, and GAPDH (loading control). B Jeko cells were synchronized with Palbociclib (500 nM) for 24 h and washed from drug. Cells were either treated with DMSO or TAK-981 immediately after Palbociclib washout (t = 0) or prior to the start of G2M (17 h) (Top) Schematic showing the time of addition of TAK-981 for each experimental condition. (Bottom) DNA profiles as a function of time. C (Top) Jeko cell were synchronized with Palbociclib (500 nM, 24 h) followed by drug washout and treatment with nocodazole (50 ng/mL) either in the presence of DMSO or TAK-981 (100 nM) for 24 h. Both drugs were then washed out and cells were collected every 2 h for cell cycle analysis and for protein for SUMOylation levels. (Bottom) Jeko cell were synchronized with Palbociclib (500 nM, 24 h) followed by drug washout and treatment with nocodazole (50 ng/mL) for 24 h. Cells were then treated with either DMSO or TAK-981 for 3 h. Cells were then washed from both drugs and treated with either DMSO or TAK-981 and cells were collected every 2 h for cell cycle analysis and for protein for SUMOylation levels

Article Snippet: For SAE1 knockdown, after lentiviral transduction from viruses obtained from transfection of SAE1 human shRNA plasmids (Origene, Cat#TL315567), cells were GFP sorted 1 day after transfection and downstream assays were carried out after three days from sorting.

Techniques: Inhibition, Conjugation Assay, Staining, Control, Cell Cycle Assay

Journal: Experimental Hematology & Oncology

Article Title: A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells

doi: 10.1186/s40164-022-00293-y

Figure Lengend Snippet: Sumoylation is significantly upregulated in MCL cells and is essential for survival. A B-cells were isolated from normal peripheral blood and activated for 3 days (See ). Lysates were prepared and blotted for the indicated proteins. B Dot plots of the relative expression levels of UBA2 (SAE2), SAE1, and UBE2I (UBC9) within different cell subtypes from peripheral blood samples from four patients with MCL with leukemic disease. C MCL cell lines (left, n = 8) and primary MCL cells isolated from the peripheral blood of patients with leukemic disease (right, n = 5) and normal donor resting B-cells were blotted for the indicated proteins. D Survival curves of MCL patients (n = 102) stratified by the upper and lower 50 th percentile of expression of SAE1, SAE2, and UBC9 using the R2 Genomic analysis and visualization platform. E Z-138 (left) UPN-1 cells (middle) or Jeko cells (right) were transduced with lentiviruses containing short hairpins targeting SAE2. After 2 days of puromycin selection, lysates were prepared and blotted for SAE1, SAE2, UBC9, SUMO1 or SUMO2. Three days after removal from selection, total viable cells were quantified. (n = 3 independent experiments for each, * p < .05)

Article Snippet: For SAE1 knockdown, after lentiviral transduction from viruses obtained from transfection of SAE1 human shRNA plasmids (Origene, Cat#TL315567), cells were GFP sorted 1 day after transfection and downstream assays were carried out after three days from sorting.

Techniques: Isolation, Expressing, Transduction, Selection

Journal: Experimental Hematology & Oncology

Article Title: A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells

doi: 10.1186/s40164-022-00293-y

Figure Lengend Snippet: Loss of sumoylation results in mitotic dysregulation with accumulation of polyploid cells A (left). Volcano plot of genes with a statistically significant association with SAE1 and SAE2 (FDR < 0.01). (right) DAVID analysis was performed on the statistically significant gene list. The top gene groups and enrichment scores are shown. B (right). Normal and activated B-cells and MCL cell lines were treated with DMSO, 50 nM or 100 nM of TAK-981 and cells were collected daily. Cells were fixed, stained with PI and cell cycle analysis was performed. Representative DNA profiles are shown for the indicated cell lines and primary samples treated with 100 nM of TAK-981. Quantification of 2n cells relative to DMSO control (top, right) at 24 h and percentage polyploid cells (bottom, right) after 48 h of TAK-981 treatment at the indicated concentrations (n = 3 independent experiments per cell line). C Primary MCL samples were treated and analyzed as in B (n = 3 independent experiments per sample). D (Left) The indicated cell lines were treated with palbociclib (200 nM) for 24 h and then released into DMSO, palbociclib, TAK-981 or palbociclib + TAK-981. (right top) Percentage viable cells was quantified after 72 h from the time of release from the initial palbociclib treatment (n = 3 independent experiments for each cell line, * p < 0.05). (right bottom) Western blot confirming equivalent losses of sumoylated proteins with TAK-981 in the presence or absence of TAK-981

Article Snippet: For SAE1 knockdown, after lentiviral transduction from viruses obtained from transfection of SAE1 human shRNA plasmids (Origene, Cat#TL315567), cells were GFP sorted 1 day after transfection and downstream assays were carried out after three days from sorting.

Techniques: Staining, Cell Cycle Assay, Control, Western Blot

Journal: Experimental Hematology & Oncology

Article Title: A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells

doi: 10.1186/s40164-022-00293-y

Figure Lengend Snippet: Inhibition of SUMO conjugation during S phase is required for proper mitotic division in MCL cells A Jeko (left) or Z-138 (right) cells were synchronized with palbociclib (500 nM) and treated with either DMSO or TAK-981 (100 nM) and lysates were prepared at the indicated time points after washout. The corresponding phase of the cell cycle based on the DNA profiles obtained from fixed, PI stained cells from each of the time points is shown. Lysates were blotted for SUMO1, SUMO2/3, SAE1, SAE2, UBC9, and GAPDH (loading control). B Jeko cells were synchronized with Palbociclib (500 nM) for 24 h and washed from drug. Cells were either treated with DMSO or TAK-981 immediately after Palbociclib washout (t = 0) or prior to the start of G2M (17 h) (Top) Schematic showing the time of addition of TAK-981 for each experimental condition. (Bottom) DNA profiles as a function of time. C (Top) Jeko cell were synchronized with Palbociclib (500 nM, 24 h) followed by drug washout and treatment with nocodazole (50 ng/mL) either in the presence of DMSO or TAK-981 (100 nM) for 24 h. Both drugs were then washed out and cells were collected every 2 h for cell cycle analysis and for protein for SUMOylation levels. (Bottom) Jeko cell were synchronized with Palbociclib (500 nM, 24 h) followed by drug washout and treatment with nocodazole (50 ng/mL) for 24 h. Cells were then treated with either DMSO or TAK-981 for 3 h. Cells were then washed from both drugs and treated with either DMSO or TAK-981 and cells were collected every 2 h for cell cycle analysis and for protein for SUMOylation levels

Article Snippet: For SAE1 knockdown, after lentiviral transduction from viruses obtained from transfection of SAE1 human shRNA plasmids (Origene, Cat#TL315567), cells were GFP sorted 1 day after transfection and downstream assays were carried out after three days from sorting.

Techniques: Inhibition, Conjugation Assay, Staining, Control, Cell Cycle Assay

Journal: Experimental Hematology & Oncology

Article Title: A sumoylation program is essential for maintaining the mitotic fidelity in proliferating mantle cell lymphoma cells

doi: 10.1186/s40164-022-00293-y

Figure Lengend Snippet: Sumoylation is significantly upregulated in MCL cells and is essential for survival. A B-cells were isolated from normal peripheral blood and activated for 3 days (See ). Lysates were prepared and blotted for the indicated proteins. B Dot plots of the relative expression levels of UBA2 (SAE2), SAE1, and UBE2I (UBC9) within different cell subtypes from peripheral blood samples from four patients with MCL with leukemic disease. C MCL cell lines (left, n = 8) and primary MCL cells isolated from the peripheral blood of patients with leukemic disease (right, n = 5) and normal donor resting B-cells were blotted for the indicated proteins. D Survival curves of MCL patients (n = 102) stratified by the upper and lower 50 th percentile of expression of SAE1, SAE2, and UBC9 using the R2 Genomic analysis and visualization platform. E Z-138 (left) UPN-1 cells (middle) or Jeko cells (right) were transduced with lentiviruses containing short hairpins targeting SAE2. After 2 days of puromycin selection, lysates were prepared and blotted for SAE1, SAE2, UBC9, SUMO1 or SUMO2. Three days after removal from selection, total viable cells were quantified. (n = 3 independent experiments for each, * p < .05)

Article Snippet: For SAE1 knockdown, after lentiviral transduction from viruses obtained from transfection of SAE1 human shRNA plasmids (Origene, Cat#TL315567), cells were GFP sorted 1 day after transfection and downstream assays were carried out after three days from sorting.

Techniques: Isolation, Expressing, Transduction, Selection

Journal: The Journal of Biological Chemistry

Article Title: Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites

doi: 10.1074/jbc.RA120.013780

Figure Lengend Snippet: A microscopy-based screen reveals SUMO-dependent regulation of Pol η association with DNA damage sites. A, experimental set-up of high-throughput microscopy-based screen for ubiquitin and UBL signaling network components regulating Pol η interaction with sites of cisplatin-induced DNA damage. See text for details. B, results of the screen outlined in A . Scatter plot shows ranked Z-scores of cisplatin-induced GFP-Pol η foci counts of all siRNAs in the library (2 siRNAs per gene), determined by QIBC analysis. Controls (RAD18-RAD6 and USP1) are indicated in green and red , respectively. SUMO E1 (SAE1 and UBA2) and E2 (UBC9) enzymes are indicated in orange . See also Table S1 . C, workflow of primary and validation screens, and hit selection. See also Table S2 . D, results of the validation screen analyzing GFP-Pol η foci count in U2OS/GFP-Pol η cells transfected with the indicated siRNAs, exposed to cisplatin for 6 h, and fixed 16 h later and quantified using QIBC analysis (mean ± S.D.; n = 3 independent experiments; ≥294 cells quantified per condition). E, results of validation screen analyzing kinetics of GFP-Pol η foci formation in cells treated as in D (mean ± S.D.; n = 3 independent experiments; ≥3,000 cells quantified per condition). F, representative images of endogenous Pol η foci formation in U2OS cells transfected with the indicated siRNAs and exposed to UV. Scale bar , 10 μm. G, immunoblot analysis of chromatin-enriched fractions of U2OS cells treated as in F. H, U2OS/GFP-Pol η cells were preincubated or not with SUMOi for 30 min, exposed to UV (20 J/m 2 ), and collected 6 h later. The sum of GFP-Pol η foci intensity per nucleus was quantified by QIBC (mean ± S.E.M.; n = 3 independent experiments; ≥7,482 cells quantified per condition). I, representative images of endogenous Pol η foci formation in U2OS, hTert RPE-1, and MRC5 cells treated as in H. Scale bar , 10 μm.

Article Snippet: Antibodies used in this study included: Actin (M1501; Millipore), CPD (D194-1; MBL International), HA (F-7, sc-7392; Santa Cruz Biotechnology), MCM6 (C-20, sc-9843; Santa Cruz Biotechnology), PCNA (PC-10, sc-56; Santa Cruz Biotechnology), Pol η (A301–231A; Bethyl Laboratories), PIAS1 (ab77231; Abcam), PIAS4 (D2F12, 4392S; Cell Signaling Technology), RAD18 (ab57447; Abcam), SAE1 (A302-923A; Bethyl Laboratories), SUMO1 (ab32058; Abcam), SUMO2/3 (ab3742; Abcam), UBA2 (SAE2) (A302–925A; Bethyl Laboratories), and UBC9 (ab21193; Abcam).

Techniques: Microscopy, High Throughput Screening Assay, Ubiquitin Proteomics, Biomarker Discovery, Selection, Transfection, Western Blot

Journal: Nature Communications

Article Title: Myc inhibition is effective against glioma and reveals a role for Myc in proficient mitosis

doi: 10.1038/ncomms5632

Figure Lengend Snippet: ( a ) Western blot analysis of samples from GBM cells shows that Omomyc causes a decrease in SAE1 expression in U87MG but not U373MG cells. GBM cells with a lentiviral Omomyc expression cassette were treated with doxycycline for 6 days to induce Omomyc expression. Two independent sets of samples are shown for each of the GBM cell lines. ( b ) Western blot analysis of GBM samples shows that Omomyc expression triggers an increase in PTP1B phosphorylation in U373MG cells. GBM cells with a lentiviral Omomyc expression cassette were treated with doxycycline for 6 days to induce Omomyc expression. A Taxol control was used to indicate the typical phosphorylation-induced mobility shift of PTP1B during mitotic catastrophe. Taxol-treated control samples are nonadjacent bands from the same western blot.

Article Snippet: Protein extracts were run on 10% or 12% precast gels (Life Technologies), transferred to PVDF membranes (Millipore) and incubated with antibodies against SAE1 (Novus Biologicals, 1:1,000), PTP1B (BD Biosciences, 1:2,000), FLAG (Sigma, 1:4,000) or actin (Sigma, 1:200,000).

Techniques: Western Blot, Expressing, Phospho-proteomics, Control, Mobility Shift

Journal: Clinical and experimental rheumatology

Article Title: Distinct dermatomyositis populations are detected with different autoantibody assay platforms

doi:

Figure Lengend Snippet: Comparison of assay data

Article Snippet: cDNAs encoding full-length human MDA5, NXP2, SAE1 and PM-Scl were purchased (OriGene, Rockville, MD).

Techniques: Enzyme-linked Immunosorbent Assay