Fig 2: hSSB1 is SUMOylated by SUMO3. a Forty-eight hours after HEK293T cells were cotransfected with FLAG-hSSB1 and HA-SUMO1-3, the cells were lysed and the proteins analyzed by western blotting or immunoprecipitation (IP) using an anti-FLAG antibody followed by western blotting. WCL whole cell lysate. b Forty-eight hours after HEK293T cells were cotransfected with FLAG-hSSB1 and His-SUMO3, the cells were lysed. After sonication, the lysates were incubated with nickel-nitrilotriacetic acid (Ni-NTA) beads, and the pulled down proteins were analyzed by western blotting. c HEK293T cells treated with or without etoposide were lysed in RIPA-SDS lysis buffer, immunoprecipitated with SUMO2/3 affinity beads and the proteins, analyzed by western blotting. d Twenty-four hours after HEK293T cells were cotransfected with FLAG-hSSB1 and HA-SUMO3, the cells were treated with or without 100 µM etoposide, as indicated, for 24 h, and then the cells were lysed and the proteins analyzed as in a. e HEK293T cells cotransfected with FLAG-hSSB1 and HA-SUMO3 were treated with etoposide for the indicated times before harvesting, 48 h post transfection, and then the cells were lysed and the proteins analyzed as in a. f HEK293T cells with stably knocked out UBC9 were cotransfected with FLAG-hSSB1 and HA-SUMO3 for 24 h, and then treated with 100 µM etoposide for 24 h, as shown in a. g, h HEK293T cells were transfected with the indicated FLAG-hSSB1 plasmids with HA-SUMO3 and, after 24 h, were treated with 100 µM etoposide, as indicated, for 24 h. Then, the cells were lysed and the proteins analyzed by western blotting or IP using the anti-FLAG antibody followed by western blotting

Fig 3: Adipocyte-specific Ube2i deletion impairs WAT expansion in male and female mice. (A) Body weight and (B) WAT weights (g) in combined male and female mice at 7 (n = 5–9), 30 (n = 4/group), 60 (n = 4/group), and 180 (n = 23–24/group) days of age for Ube2ifl/fl (gray) and Ube2ia-KO (purple) mice. Data are presented as mean +/- SEM; *p < 0.05. (C) Relative gene expression by qPCR in gWAT (left, yellow) and iWAT (right, cyan) from Ube2ia-KO (purple) and Ube2ifl/fl control (gray) mice for adipocyte maturation, lipid metabolism, inflammatory, and senescence genes represented as a heatmap of z-scores. Gray heatmap squares indicate outliers excluded based on Grubbs' test. (D) Representative H/E stained gWAT and iWAT sections from one-month-old Ube2ia-KO and Ube2ifl/fl control mice. Scale bar 100 µm. (E)Ube2ia-KO and Ube2ifl/fl control mice were weighed for up to 23 weeks in males (n = 12–14/group, mean +/- SD) and females (n = 6–8/group, mean +/- SD). (F) Assessment of fat and lean mass (% body weight (left) and in grams (right); male n = 11–15/group, female n = 5–7/group). (G) Tissue weights (% body weight; male n = 15-16/group, female n = 8/group) at necropsy. (H) Corresponding necropsy images from six-month-old Ube2ifl/fl and Ube2ia-KO mice. Images of excised tissues demonstrate gross morphological increases in liver size, reductions in iWAT and gWAT, and lighter coloring of liver and BAT. (E–H) Gray = Ube2ifl/fl male and female controls, blue = male Ube2ia-KO, red = female Ube2ia-KO. Data are presented as mean +/- SEM; *p < 0.05.

Fig 4: Cas9 enzyme is subject to modification by SUMO1 and SUMO2/3.(A) Immunoprecipitation of Cas9 expressed in HEK293 cells reveals Cas9 conjugation by SUMO1 and SUMO2/3 peptides. HEK293 cells were transfected with doxycycline (Dox)-inducible FLAG-tagged Cas9 (from Streptococcus pyogenes) along with GFP-tagged SUMO paralogs. Pull-down of FLAG-–Cas9 was followed by Western blot analysis with SUMO1, SUMO2/3, or GFP antibodies, which yielded a smear pattern corresponding to sumoylated Cas9 (Cas9–SUMO, highlighted by brackets). Negative controls are shown in Fig 2B (pull-down with a nonspecific IgG, also pull-down from cells expressing GFP-SUMO only) and in Fig S1A. (B) Verification of Cas9 sumoylation by His pull-down. HEK293 cells were transfected with FLAG–Cas9 along with histidine-tagged SUMO paralogs (His-SUMO1 or His–SUMO2/3). Nickel-purified His-SUMO conjugates were probed with a FLAG antibody to detect sumoylated Cas9 forms (highlighted by asterisks; a small fraction of unmodified Cas9 adsorbs non-specifically to beads [arrowhead]). (C) Immunoprecipitation of Cas9 expressed in HEK293 cells reveals Cas9 conjugation by endogenous SUMO1 and SUMO2/3. HEK293 cells were transfected with doxycycline (Dox)-inducible FLAG-tagged Cas9 only. (D) Proximity ligation (Duolink) assays probe Cas9 modification by endogenous SUMO1 and SUMO2/3 peptides, as well as physical interaction with the UBC9 E2 SUMO conjugating enzyme in HEK293 cells stably expressing Streptococcus pyogenes Cas9. Z-stack projections are shown. Nuclei were stained with DAPI. Quantifications of positive Duolink signals are shown in the graph. n > 25 cells per experiment. Data represent mean value from two experiments per condition, including the negative controls using a single antibody of a given Duolink pair, ± SEM (****P < 0.0001, unpaired t test). Representative confocal images of the negative controls are shown in Fig S1C. For simplicity, P-values between the following groups were not included in the graph: PCas9-SUMO1 & Cas9 < 0.0001, PCas9-SUMO1 & SUMO1 < 0.0001, PCas9-SUMO2/3 & Cas9 < 0.0001, PCas9-SUMO2/3 & SUMO2/3 < 0.0001, PCas9-Ubc9 & Cas9 < 0.0001, PCas9-Ubc9 & Ubc9 < 0.0001.

Fig 5: Post-Translational Modifications on UBC9 in U2OS cells Identified by IP and LC-MS/MS. (a) UBC9 is a heavily post-translationally modified protein as shown by the distribution of SUMOylation, Ubiquitylation and Acetylation sites identified on UBC9 from our IP and LC-MS/MS experiments. (b) Modified sites depicted on the UBC9 crystal structure (3UIP), using the color scheme from (a). (c) Model of the interaction brought about by the SUMOylation of UBC9 at Lys-49 and PML as an example of a protein with both SUMOylated site and SIM (SUMO interacting motif). (d) Quantification for the colocalization of UBC9 and PML were obtained from immunofluorescence analyses using monoclonal mouse anti-Flag and rabbit anti-PML antibodies in U2OS cells stably expressing SUMO3m with wild type UBC9 (F-Ubc9 WT) or its K49R variant (F-Ubc9K49R) and with an empty vector (Vector) or a vector expressing RAS. Cells were fixed for immunofluorescence ten days after transduction. (e) Quantification of the number of PML-NBs in U2OS cells when expressing SUMO3m with wild type UBC9 (F-Ubc9 WT) or its K49R variant (F-Ubc9K49R) and with an empty vector (Vector) or a vector expressing RAS.