Fig 2: A mechanism of how SUMOylation is involved in CSC function.(a) Overexpression of Oct-1 partially compensated for the reduction of CSC frequency by SAE2 knockdown in HT29 cells as determined by LDA. Stable cell lines were generated with lentivirus expressing pLenti CMV-hygro empty vector (EV) or pLenti CMV-Flag-Oct-1 in HT29 shCtrl and shSAE2 cells as shCtrl+EV, shSAE2+EV and shSAE2+Oct-1. (b) Representative western blot of the stable lines to confirm expression with Oct-1 and Flag-tag antibodies; GAPDH, loading control. (c) Overexpression of Oct-1 in SAE2 or Ubc9 knockdown cells restored ALDH+ cells population in HT29 cells as measured by FACS analysis using the AldeFluor kit. HT29 cells were transfected with control non-targeting siRNA (SiCtrl), SAE2-targeting siRNA (SiSAE2) or Ubc9-targeting siRNA (SiUbc9) followed by Flag-Oct-1 plasmid or control empty vector transfection. After 3 days, cells were collected for FACS analysis using the AldeFluor kit. (d) Representative western blot of the samples from c; quantification of the ALDH1A1 band intensity is shown on the bottom. (e) Knockdown of Ubc9 reduced CSC frequency, as shown by LDA using spheroid formation using HT29 stable cell lines expressing two different UBC9-targeting shRNA (shUBC9#1 and shUBC9#2). (f) Schematic diagram showing the mechanism of how SUMOylation regulates CSCs through Oct-1, TRIM21 and IRF1. *P<0.05, **P<0.01 and ***P<0.001.

Fig 3: PIAS3 association with ESR1 via NR2E3A-B. MCF-7 breast cancer cells were transiently transfected with NCOA1, 2 and 3 specific siRNA Smart Pool or control siLuc. Protein lysates were isolated from indicated samples and used for Western blot with indicated antibodies (A) and 30 ng of total RNA from transfected cell lines were analysed by qRT-PCR using gene-specific primers as indicated (B). Student t-test (two-tailed) was applied to estimate the significance of gene expression changes: ***p < 0.005, **p < 0.01 and *p < 0.05.C-D. MCF-7 cells were transiently transfected with siPIAS3 or siLuc. Total RNA or protein from indicated cell extracts was analysed by qRT-PCR (C) or Western blot (D) to detect the indicated mRNA or protein expression levels.E. MCF-7 cells were transiently transfected with siESR1 or siLuc. Protein from indicated cell extracts was analysed by Western blot to detect the indicated protein expression levels.F. ESR1 promoter construct was transfected with indicated constructs after indicated siRNA was transfected with MCF-7 cells, and the cells were harvested for luciferase assay. Values indicated relatively normalized luciferase activity. Western blot shows silencing efficiency of UBC9.G. Co-IP of NR2E3 with PIAS3 analysed by Western blotting from MCF-7 cells.H. GST-NR2E3 was incubated with His-PIAS3 for 2 h at 4°C and then isolated from reaction mixture by an immobilized nickel resin. The resulting precipitates were subjected to immunoblot analysis to NR2E3 or PIAS3.I. ChIP assay was done in MCF-7 with PIAS3 or NR2E3 antibodies. Recruitment of PIAS3 to the ESR1 promoter was analysed using a primer specific to the ESR1 promoter. IgG was used as an internal control.J. After MCF-7 cells were transiently transfected with siPIAS3 or siLuc., cells were used for ChIP assay.

Fig 4: Colorectal cancer stem cells have higher SAE2 and global SUMOylation levels than non-cancer stem cells.(a) Representative colony formation assay of ALDH+ and ALDH- cells isolated from HT29 cells (scale bar, 50 µm). (b) Tumour development of ALDH+ and ALDH- cells isolated from HT29 that were injected in NSG mice (500 cells per mice) subcutaneously. (c) Cell viability assay of ALDH+ and ALDH- cells after irradiation (4 Gy) (***P<0.001). (d) Western blot showing ALDH, SAE2, Ubc9 and global SUMOylation (SUMO-2,3) level of ALDH+ and ALDH- cells isolated from colorectal cancer cell line HT29 and primary colon cancer PDX tumour tissue (386532 and 354313 correspond to PDX tissues from different patient specimen); GAPDH, loading control.

Fig 5: K107R induces gene expression in an adipocyte-autonomous manner. (A) Comparison of gene regulation by K107R and rosiglitazone in vivo and in vitro. For the in vivo analysis, RNA was prepared from iWAT and eWAT from WT and K107R mice administered rosiglitazone or vehicle by oral gavage for 7 d. For the in vitro analysis, RNA was prepared from WT and K107R stromal vascular cells derived from iWAT and differentiated for 8 d using the insulin/dexamethasone/IBMX mixture and subsequently incubated for 48 h with either rosiglitazone or vehicle. Gene expression was measured by qPCR. Cycle threshold (Ct) values are shown for WT/vehicle conditions. (B) Immunoblotting for SUMO1-conjugated PPAR? (SUMO1) and total PPAR? following PPAR? immunoprecipitation (IP) (Top Two Rows) and total PPAR?, UBC9, and ß-actin in total cell extract (Bottom Three Rows) from WT and K107R adipocytes treated with a control or UBC9 siRNA. (C) Quantification of Fgf21 and Elovl3 gene expression by qPCR using RNA prepared from WT and K107R adipocytes exposed to either the UBC9 or control siRNA. Ct values are shown for WT/control siRNA conditions. For B and C, WT and K107R stromal vascular cells were treated with control or UBC9 siRNA for 2 d in growth medium. The cells were subsequently incubated for 2 d in differentiation medium containing insulin, dexamethasone, IBMX, and rosiglitazone, followed by 2 d in medium containing only insulin and rosiglitazone. *P < 0.05; **P < 0.01; ***P < 0.001.