Journal: Molecular Cell

Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

doi: 10.1016/j.molcel.2020.11.029

Figure Lengend Snippet: RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see ).

Article Snippet: SUMO E1 inhibitor (ML-792; Medkoo Biosciences) was supplemented to egg extracts at the final concentration of 50 μM.

Techniques: Incubation, Ubiquitin Proteomics, DNA Synthesis

Journal: Molecular Cell

Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

doi: 10.1016/j.molcel.2020.11.029

Figure Lengend Snippet:

Article Snippet: SUMO E1 inhibitor (ML-792; Medkoo Biosciences) was supplemented to egg extracts at the final concentration of 50 μM.

Techniques: Ubiquitin Proteomics, Recombinant, Mutagenesis, Staining, Magnetic Beads, Transfection, Sequencing, Software

Journal: Cancers

Article Title: SUMOylation Is Associated with Aggressive Behavior in Chondrosarcoma of Bone

doi: 10.3390/cancers13153823

Figure Lengend Snippet: The SUMO E1 inhibitor ML792 blocks SUMO conjugation. ( A ) Cartoon of the function of the SUMO E1 inhibitor ML792. ( B ) Western blot analysis of SUMO2/3 and ubiquitin levels of the chondrosarcoma cell lines CH2879, JJ012, and NDCS1 treated with the indicated concentrations of the SUMO E1 inhibitor ML792 for 4 h compared to solvent control (DMSO 0.1%). Ponceau S staining was used as loading control. ( C ) Quantitative analysis of SUMO2/3 and ubiquitin conjugation of the corresponding western blots shown in B. Data represent mean with standard deviation ( n = 3).

Article Snippet: ML792 [ ] was obtained from Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Tokio, Japan), and was dissolved in DMSO for in vitro usage.

Techniques: Conjugation Assay, Western Blot, Ubiquitin Proteomics, Solvent, Control, Staining, Standard Deviation

Journal: Cancers

Article Title: SUMOylation Is Associated with Aggressive Behavior in Chondrosarcoma of Bone

doi: 10.3390/cancers13153823

Figure Lengend Snippet: Inhibition of SUMO E1 via ML792 results in decreased proliferative capacity and cell viability. ( A ) Two-dimensional (2D) colony-formation assay (crystal violet). CH2879, JJ012, and NDCS1 cells were treated with ML792 cells using single treatment or repetitive treatment on day 1, 4, 7, and 10 after seeding. Colony-formation was quantified via measuring crystal violet staining. Data represent mean with standard deviation ( n = 3). ( B ) Viability assay using PrestoBlue. Chondrosarcoma cell lines were treated with ML792 and incubated for 3 days. Relative cell viability is represented as mean with standard deviation ( n = 3). ( C ) Expression levels of c-MYC, UBA2, UBC9 and conjugation of SUMO2/3 in lysates of untreated chondrosarcoma cell lines from C. Ƴ -tubulin was used as loading control. Single representative images are shown ( n = 3). Whole blots including intensity readings can be found in ( D ) Table displaying specifics of the cell lines from B including IC50 (nM) values. ( E ) Boxplots to compare IC50 values of dedifferentiated ( n = 3) and grade 2/3 ( n = 4) chondrosarcoma cell lines. Significance was calculated with a two-tailed t -test *: p < 0.05.

Article Snippet: ML792 [ ] was obtained from Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Tokio, Japan), and was dissolved in DMSO for in vitro usage.

Techniques: Inhibition, Colony Assay, Staining, Standard Deviation, Viability Assay, Incubation, Expressing, Conjugation Assay, Control, Two Tailed Test

Journal: Cancers

Article Title: SUMOylation Is Associated with Aggressive Behavior in Chondrosarcoma of Bone

doi: 10.3390/cancers13153823

Figure Lengend Snippet: SUMO E1 inhibition leads to G2/M arrest and chromosome bridge formation. ( A ) Cell cycle analysis by flow cytometry of chondrosarcoma cell lines treated with ML792 for 24 h. Western blot analysis confirmed the inhibition of SUMO conjugation by the SUMO E1 inhibitor ML792. ( B ) Data representation of A in bar-graphs indicating G1, S, and G2/M pool of the cell lines for 0 or 24 hours of treatment as mean with standard deviation ( n = 3). ( C ) Immunofluorescence images of a-synchronous CH2879, NDCS1, and JJ012 cells treated with ML792 for 24 h. Cells were stained with Hoechst to visualize DNA. Green arrows indicate the location of chromosome bridges. Insert: magnification of a chromosome bridge. Scale bars represent 10 µm. ( D ) Quantification of chromosome bridges in CH2879 cells upon treatment with the indicated concentration of the SUMO E1 inhibitor (ML792) for 24 and 48 h. For each replicate 15 images (approximately 200 cells total) per condition were analyzed. Data represent mean with standard deviation ( n = 3). ( E ) Quantification of micronuclei present in CH2879 cells upon treatment with the indicated concentration of the SUMO E1 inhibitor (ML792) for 24 and 48 h. For each replicate 15 images (approximately 200 cells total) per condition were analyzed. Data represents mean with standard deviation ( n = 3).

Article Snippet: ML792 [ ] was obtained from Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Tokio, Japan), and was dissolved in DMSO for in vitro usage.

Techniques: Inhibition, Cell Cycle Assay, Flow Cytometry, Western Blot, Conjugation Assay, Standard Deviation, Immunofluorescence, Staining, Concentration Assay