Fig 1: HK2 can be SUMOylated at Lys 315 and Lys 492, and de-SUMOylated by SENP1.a Coimmunoprecipitation of HK2 and UBC9 protein in HEK293T cells transfected with Flag-Ubc9 and HK2-HA plasmids. b SUMO1 and SUMO2 ligation with HK2 protein in HEK293T cells transfected with HK2-HA, UBC9, and Flag-SUMO1/Flag-SUMO2. c Endogenous HK2 SUMOylation was detected by immunoprecipitation with IgG or anti-HK2 antibody and then western blotting with anti-SUMO1 or anti-SUMO2/3 antibodies. d Sequence alignment of HK2 homologs in various species. The potential SUMOylation site is denoted in red. e 293T cells were transfected with HA-tag HK2 wild-type (WT), HK2-K315R, HK2-K492R, or HK2-DKR with or without His-SUMO1 and UBC9. Cell lysates were prepared for precipitation with Ni2+-NTA resin, followed by western blotting with indicated antibodies. f A reciprocal immunoprecipitation assay was performed with IgG or SUMO1 antibody for the lysates of PC3 cells, and then western blotting with HK1 and HK2 antibodies. g SENP1 depletion enhanced HK2 SUMOylation. UBC9, SENP1, SENP2, or SENP3 was stably knocked down by shRNA in PC3 cells. Cell lysates were prepared for precipitation with HK2 antibody-conjugated protein A/G agarose beads, and SUMOylation was detected by SUMO1 antibody. h Coimmunoprecipitation of HK2 and SENP1 protein in HEK293T cells transfected with Flag-SENP1 and HK2-HA plasmids. i Overexpression SENP1 decreased while UBC9 increased SUMOylation of HK2. Flag-UBC9 or Flag-SENP1 was transfected into PC3 cells, followed by detection of HK2 SUMOylated bands with SUMO1 antibody. Source data are provided as a Source Data file.
Fig 2: Generation of forebrain-specific SENP2 cKO mice. a The schematic diagram of SENP2 cKO mouse generation. b Genotyping identification of conditional knockouts by PCR. c Confocal microscopy photomicrographs showing double immunostaining of CaMKIIα (green) and SENP2 (red) in excitatory neurons of 6-week-old cKO and littermate control mice. There is significantly less SENP2 positive excitatory neurons in cortex (Ctx) and hippocampus (Hip), but not in cerebellum (Ceb) brain slices. Scale bar = 50 μm. d In 6-week-old cKO mice, As detected by western blots, the SENP2 protein level is significantly reduced in the Ctx and Hip, but not in the Ceb of 6-week-old SENP2 cKO mice (left panel). Quantification of the Western blots is shown in the right panel. e As detected by qPCR, the SENP2 mRNA level is significantly reduced in Ctx and Hip, but not in the Ceb of 6-week-old cKO mice. All data are presented as mean ± SEM. Ctrl (Control): n = 3; cKO (SENP2 conditional knockout): n = 3. Statistical analysis performed with two-way ANOVA followed by Bonferroni’s post-hoc. *p < 0.05, **p < 0.01, ***p < 0.001 compared with littermate controls
Fig 3: Overexpression of ORC2-SUMO2 fusion protein partially reversed phenotype of cells stably expressing PIAS4 shRNA or SENP2(A) Various U2OS cells lines as indicated were subjected to western blot analysis with indicated antibodies. (B) 2X105 U2OS cells stably expressing control shRNA, control shRNA/ORC2-SUMO2, PIAS4 shRNA or PIAS4 shRNA/ORC2-SUMO2 as indicated (upper panel) or EV, EV/ORC2-SUMO2, SENP2 or SENP2/ORC2-SUMO2 as indicated (lower panel) were plated and cell number was counted every day for five days. The graph shows the average of three independent experiments; mean ± SD. *, p < 0.05; **, p < 0.01. (C and D) 1000 U2OS cells stably expressing control shRNA, PIAS4 shRNA or PIAS4 shRNA/ORC2-SUMO2 (C) or EV, SENP2 or SENP2/ORC2-SUMO2 (D) were seeded per well of a six-well plate. After incubation for 10–14 days, cells were stained with crystal violet (upper panel). Analysis of colony formation rates of cells (lower panel). Data are means ± SD of three independent experiments. *, p < 0.05; **, p < 0.01. (E) U2OS cells were transient transfected with Flag tagged ORC2-SUMO2. Western blot and FACS were performed to detect expression of Flag-ORC2-SUMO2 protein and cell cycle profile, respectively. (F and G) Cell cycle profiles of cells stably expressing control shRNA, PIAS4 shRNA or PIAS4 shRNA/ORC2-SUMO2 (F) or EV, SENP2 or SENP2/ORC2-SUMO2 (G) were analyzed by FACS.
Fig 4: Cell cycle defects induced by SENP2 silencing do not activate a DNA damage response.A Immunofluorescence for 53BP (green) in primary keratinocytes 2 or 5 days post-infection (dpi). Nuclear DNA labelled with DAPI in blue. Scale bar 50 μm. Bar histogram: percent of positive 53BP cells, 2 or 5 days post-infection with shSP2, as indicated (n = 71-381 cells). B Western blotting for the expression of p51, p21 or γH2AX in primary keratinocytes, 5 days post-infection. GAPDH (GDH) as loading control. C Immunofluorescence for RAD51 in RPE-1 cells 3 days post-transfections with CT or siSP2 as indicated. Scale bar 20 μm. Bar histogram: percent of positive RPE-1 cells with multiple foci of RAD51, 3 days post-transfection with siSP2 (n = 65–69 cells). D Western blotting for the expression of FOXM1 (FM1) and Cyclin A (CA) in RPE-1 cells, 3 days post-transfections with siSP2. GAPDH (GDH) as loading control.
Fig 5: Depletion of SENP2 induces cellular polyploidy and other cell cycle defects in primary keratinocytes.Results were obtained 5 days post-infections. A, B. Bar histograms displaying the percent of cells in G2/M relative to G1 population or polyploid cells, as indicated (n = 3). C Representative flow cytometry histograms of cell cycle profiles labelled with Propidium Iodide (PI). D, E Bar histograms displaying the percent of positive cells for BrdU staining, and for BrdU negative cells in S phase, as indicated (n = 3). F Representative flow cytometry dot plot for BrdU incorporation along cell cycle phases. G Western blotting analyses of the expression of pRB, Cyclin A (CA), Cyclin E (CE), Cyclin B (CB), CDK1, FOXM1 (FM1), CHK1 and WEE1. GAPDH (GDH) as loading control. H Double immunofluorescence for Cyclin B (green) and Cyclin A (red) labelling. Nuclei labelled with DAPI in blue. Scale bar 50 μm.
Supplier Page from Abcam for Anti-SENP2 antibody