Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet: Inhibition of MBNL2 activated SUMOylation of KLF4 (A–D) Representative graphs of SUMO1/2/3 protein in Young vs. Aged, Control vs. H 2 O 2 , oe-NC vs. oe-MBNL2, sh-NC vs. sh-MBNL2. (E–G) Representative and statistical graphs of SENP1 and SUMO1 protein. (H–J) Representative and statistical graphs of KLF4 and SENP1 protein. (K) Representative immunoblots of KLF4-SUMOylation after incubation with sh-MBNL2 or/and H 2 O 2 in CFs ( n = 3). (L–O) Western blot analysis and quantification of TGF-β1, T-SMAD3 and P-SMAD3 protein levels (n = 3–6, data are expressed as mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01 vs. the control group, # p < 0.05, ## p < 0.01 vs. the H 2 O 2 group, & p < 0.05, && p < 0.01 vs. the H 2 O 2 +sh-MBNL2 group).

Article Snippet: Anti-SUMO1 antibody , Proteintech , Cat# 10329-1-AP; RRID:AB_2286872.

Techniques: Inhibition, Control, Western Blot, Incubation

Journal: iScience

Article Title: MBNL2 promotes aging-related cardiac fibrosis via inhibited SUMOylation of Krüppel-like factor4

doi: 10.1016/j.isci.2024.110163

Figure Lengend Snippet:

Article Snippet: Anti-SUMO1 antibody , Proteintech , Cat# 10329-1-AP; RRID:AB_2286872.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Magnetic Beads, Plasmid Preparation, Extraction, Microarray, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: SUMOylation determines the voltage required to activate cardiac I Ks channels.

doi: 10.1073/pnas.1706267114

Figure Lengend Snippet: Fig. 1. SUMO regulates IKs in neonatal mouse cardiac ventricular myocytes. nCM were identified by visualization of muscular striations and spontaneous beating. IKs was isolated with 30 μM Chromanol-293B in the bath by the pro- tocols described in the main text and Materials and Methods; biophysical pa- rameters are in Table 1 as mean ± SEM for n = 6–11 cells. Currents measured before application of Chromanol-293B and the Chromanol-293B–insensitive current are shown in Fig. S1. (A) Subunit topology of KCNQ1 (black) and KCNE1 (green) show the expected stoichiometry. (B, Upper Left) nCM photomicro- graph. (Scale bar: 50 μm.) (B, Lower Left) Representative family of currents for native IKs. (B, Right) Representative isochronal conductance–voltage relation- ships for native IKs with control solution in the pipette. (C) SENP2 mRNA level measured by qPCR from groups of 5–10 eGFP-positive nCMs: untreated control (black), SENP2-shRNA knockdown (SENP2 K.D., pink), and scramble-shRNA (gray). SENP2-shRNA reduced the level of SENP2 mRNA by 51 ± 6%, com- pared with the scramble-shRNA, (**P < 0.01). (D) iG-V relationships for native IKs with control solution in the pipette as shown in B (black dash), 1 nM SENP2 (+SENP, blue), 1 nM SUMO2 (+SUMO, red), for nCM exposed to lentivirus expressing shRNA for SENP2 knockdown (SENP2 K.D., pink) or a scramble shRNA (scramble, gray).

Article Snippet: Lentiviral particles expressing GFP and an shRNA targeting mouse SENP2 or a scrambled control (OriGene Technologies) were applied to nCM for 12 h, where indicated.

Techniques: Isolation, Control, Transferring, shRNA, Knockdown, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: SUMOylation determines the voltage required to activate cardiac I Ks channels.

doi: 10.1073/pnas.1706267114

Figure Lengend Snippet: Fig. 2. The SUMO pathway regulates human IKs in CHO cells. Human KCNQ1 and KCNE1 subunits were transfected into CHO cells, and whole-cell patch-clamp recordings were performed 24–48 h afterward, as described by the protocols in Materials and Methods. Biophysical parameters are in Table 2 (n = 6 cells, mean ± SEM). (A, Upper) iG-V relationships (filled square) for wild-type human KCNQ1 and KCNE1 with 1 nM SENP2 (+SENP, blue), control solution (black), and 1 nM SUMO2 (+SUMO, red). (A, Lower) Raw current traces for wild-type KCNQ1 and KCNE1 with 1 nM SENP2 (+SENP, blue), control solution (black), and 1 nM SUMO2 (+SUMO, red). (B, Upper) iG-V relationships for human KCNQ1-Lys424Gln and KCNE1 (open square) with 1 nM SENP2 (+SENP, blue), 1 nM SUMO2 (+SUMO, red), and control solution (black). (B, Lower) Raw current traces for KCNQ1-Lys424Gln and KCNE1, with 1 nM SENP2 (+SENP, blue), control solution (black), and 1 nM SUMO2 (+SUMO, red).

Article Snippet: Lentiviral particles expressing GFP and an shRNA targeting mouse SENP2 or a scrambled control (OriGene Technologies) were applied to nCM for 12 h, where indicated.

Techniques: Transfection, Patch Clamp, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: SUMOylation determines the voltage required to activate cardiac I Ks channels.

doi: 10.1073/pnas.1706267114

Figure Lengend Snippet: Fig. 6. In the absence of KCNE1, KCNQ1 channels carry only up to two SUMOs. KCNQ1 or KCNQ1-Lys424Gln channels were expressed in CHO cells without KCNE1 and assessed for whole-cell currents with control, 1 nM SUMO2, or SENP2 in the pipette, as per Fig. 2; FRET, as per Fig. 3; and stoichiometry by TIRFM (as per Fig. 4). Biophysical parameters and single-particle statistical analyses are summarized in Tables 2 and 3. (A, Left) iG-V relationships for wild-type human KCNQ1 channels vary with 1 nM SENP2 (+SENP, blue), control solution (black), or 1 nM SUMO2 (+SUMO, red) in the pipette, showing V1/2 of −32 ± 1 mV, −16 ± 1 mV, and −14 ± 2 mV, respectively, n = 5–6 cells each group. (A, Lower Left) Representative raw current traces for a cell with SENP2 (+SENP, blue), control solution (black), or SUMO2 (+SUMO, red). (A, Right) iG-V relationships for human KCNQ1-Lys424Gln channels with 1 nM SENP2 (+SENP, blue), 1 nM SUMO2 (+SUMO, red), and control solution (black) do not vary, showing V1/2 of −32 ± 1 mV, −32 ± 2 mV, and −31 ± 1 mV, respectively, n = 5–6 cells each group. (A, Lower Right) Representative raw current traces for a cell with SENP2 (+SENP, blue), control solution (black), or SUMO2 (+SUMO, red). (B) FRET shows assembly of KCNQ1-CFP (black bars) with YFP-SUMO2 and YFP-Ubc9 (τ = 22.7 ± 0.7 and 22.4 ± 0.4, respectively, P < 0.001) but not with linkage-incompetent YFP-SUMO291 or free YFP (τ = 10.4 ± 0.7 and 10.0 ± 0.6, respectively). In contrast, KCNQ1-Lys424Gln-CFP (open bars) did not show FRET with YFP-SUMO2, YFP-SUMO291, YFP-Ubc9, or free YFP (τ = 10.7 ± 0.8, 9.9 ± 0.7, 10.5 ± 0.6, and 10.1 ± 0.6, respectively). (C) TIRFM photobleaching shows four KCNQ1 subunits and two SUMO2 subunits in each channel complex. TIRFM single-particle statistical analysis is summarized in Table 3. (D) The cartoon indicates stoichiometry: KCNQ1 subunits are black, KCNE1 subunits are green, and SUMO2 subunits are red. The effect of KCNE1 on V1/2 without SUMOylation, inferred from study of KCNQ1-Lys424Gln channels, and when wild-type IKs channels carry four SUMO2 monomers.

Article Snippet: Lentiviral particles expressing GFP and an shRNA targeting mouse SENP2 or a scrambled control (OriGene Technologies) were applied to nCM for 12 h, where indicated.

Techniques: Control, Transferring, Single Particle

Journal: Experimental and Therapeutic Medicine

Article Title: SUMO-specific protease 2 (SENP2) functions as a tumor suppressor in osteosarcoma via SOX9 degradation

doi: 10.3892/etm.2018.6838

Figure Lengend Snippet: SENP2 was downregulated in OS samples and cell lines. SENP2 (A) mRNA and (B) protein expression in OS and adjacent normal tissues. SENP2. (C) mRNA and (D) protein expression in four OS cell lines and normal human osteoblastic hFOB1.19 cells. ***P<0.005 vs. adjacent normal tissues; ##P<0.01 and ###P<0.001 vs. hFOB1.19. SENP2, small ubiquitin-like modifier-specific protease 2; OS, osteosarcoma.

Article Snippet: KO plasmids, including SENP2 CRISPR/Cas9 KO Plasmid (h): sc-402148 and SENP2 homology directed repair (HDR) Plasmid (h): sc-402148-HDR, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Expressing, Ubiquitin Proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: SUMO-specific protease 2 (SENP2) functions as a tumor suppressor in osteosarcoma via SOX9 degradation

doi: 10.3892/etm.2018.6838

Figure Lengend Snippet: SENP2 regulates OS cell proliferation, migration and invasion. (A) SENP2 protein expression in MG63 cells with or without Flag-SENP2 overexpression. (B) Cell growth curve, (C) migration assay and (D) invasion assay for Con-MG63 and Flag-SENP2-MG63 cells. (E) SENP2 protein expression in control or SENP2 KO cells. (F) Cell growth curve, (G) migration assay and (H) invasion assay for control and SENP2 KO Hos cells. **P<0.01 and ***P<0.005 vs. CON; ##P<0.01 and ###P<0.005 vs. WT. SENP2, small ubiquitin-like modifier-specific protease 2; OS, osteosarcoma; KO, knockout; WT, wild type; Con, control.

Article Snippet: KO plasmids, including SENP2 CRISPR/Cas9 KO Plasmid (h): sc-402148 and SENP2 homology directed repair (HDR) Plasmid (h): sc-402148-HDR, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Migration, Expressing, Over Expression, Invasion Assay, Control, Ubiquitin Proteomics, Knock-Out

Journal: Experimental and Therapeutic Medicine

Article Title: SUMO-specific protease 2 (SENP2) functions as a tumor suppressor in osteosarcoma via SOX9 degradation

doi: 10.3892/etm.2018.6838

Figure Lengend Snippet: SENP2 is associated with SOX9. U2OS cells were transfected with (A) Flag-SENP2 and HA-SOX9 or (B) HA-SOX9 alone for 48 h and subjected to IP with Flag M2 beads as well as western blotting. SENP2, small ubiquitin-like modifier-specific protease 2; SOX9, Sry-box-9; HA, hemagglutinin; IP, immunoprecipitation.

Article Snippet: KO plasmids, including SENP2 CRISPR/Cas9 KO Plasmid (h): sc-402148 and SENP2 homology directed repair (HDR) Plasmid (h): sc-402148-HDR, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: Experimental and Therapeutic Medicine

Article Title: SUMO-specific protease 2 (SENP2) functions as a tumor suppressor in osteosarcoma via SOX9 degradation

doi: 10.3892/etm.2018.6838

Figure Lengend Snippet: SENP2 promotes SOX9 ubiquitination and degradation. (A) U2OS cells were transfected with the indicated plasmids for 36 h and subsequently treated with 20 µM MG132 for 4 h, following which western blotting was performed. (B) SOX9 protein expression was measured in control and SENP2 KO cells. (C) Control and SENP2 KO cells were treated with 20 µg/ml CHX for 0, 1, 2, 4 or 8 h and the protein expression of SOX9 was measured. (D) U2OS cells were transfected with the indicated plasmids and subjected to immunoprecipitation with HA-antibody and western blotting. SENP2, small ubiquitin-like modifier-specific protease 2; SOX9, Sry-box-9; KO, knockout; MUT, mutant; WT, wild type; HA, hemagglutinin.

Article Snippet: KO plasmids, including SENP2 CRISPR/Cas9 KO Plasmid (h): sc-402148 and SENP2 homology directed repair (HDR) Plasmid (h): sc-402148-HDR, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Expressing, Control, Immunoprecipitation, Knock-Out, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: SUMO-specific protease 2 (SENP2) functions as a tumor suppressor in osteosarcoma via SOX9 degradation

doi: 10.3892/etm.2018.6838

Figure Lengend Snippet: The SENP2-SOX9 axis regulates OS cell proliferation and invasion. (A) Cell growth curve and (B) migration assay for SENP2 KO cells transfected with control-shRNA or SOX9-shRNA. ***P<0.005 vs. Con. SENP2, small ubiquitin-like modifier-specific protease 2; SOX9, Sry-box-9; OS, osteosarcoma; shRNA, short hairpin RNA; Con, control.

Article Snippet: KO plasmids, including SENP2 CRISPR/Cas9 KO Plasmid (h): sc-402148 and SENP2 homology directed repair (HDR) Plasmid (h): sc-402148-HDR, were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

Techniques: Migration, Transfection, Control, shRNA, Ubiquitin Proteomics

Journal: Journal of cell science

Article Title: SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability.

doi: 10.1242/jcs.036632

Figure Lengend Snippet: Fig. 1. SUMOylation regulates Kv2.1 and native INS-1 Kv current. (A)HEK 293 cells were transfected with the indicated cDNAs. Immunoprecipitation was performed using an anti-Kv2.1 antibody and analyzed by immunoblotting using anti-Kv2.1 and anti-GFP antibodies as indicated, and identified by the green and red signals, respectively, in the colored panel. Colocalization of these is shown in yellow. (B,C)Representative current traces and current-voltage relationships from HEK 293 cells expressing Kv2.1 alone (control, black circles) or together with SUMO1-YFP (SUMO1, black triangles), SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (white circles). (D)Current-voltage relationships from HEK 293 cells expressing Kv2.1 and infused with recombinant GST (black circles), SUMO1 (black triangles) or SUMO1+Ubc9 (open triangles) peptides. (E,F)Representative current traces and current-voltage relationships from INS1 832/13 cells expressing EGFP (control, black circles), SUMO1-YFP (SUMO1, black triangles), SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (open circles). *P<0.05, **P<0.01 and ***P<0.001 compared with control; error bars, s.e.m.

Article Snippet: The human SUMO1 (untagged), Ubc9 and SENP1 constructs in the pCMV6-XL4 vector were from Origene Technologies (Rockville, MD).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Recombinant

Journal: Journal of cell science

Article Title: SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability.

doi: 10.1242/jcs.036632

Figure Lengend Snippet: Fig. 2. SUMOylation regulates native human β-cell Kv current. (A)Representative current traces from human β-cells expressing EGFP alone (control) or SUMO1-YFP (SUMO1) with Ubc9 and/or SENP1 as indicated. (B- D)Current-voltage relationships were from β-cells from three human donors expressing EGFP (black circles), SUMO1-YFP (black triangles), SUMO1- YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (open circles). (E)Same as B-D, but data from all donors was pooled. (F,G)Representative current traces and current-voltage relationships from human β-cells (two donors) infused with recombinant GST (black circles), SUMO1 (black triangles), or SUMO1+Ubc9 (open triangles). *P<0.05, **P<0.01 and ***P<0.001 compared with control; error bars, s.e.m.

Article Snippet: The human SUMO1 (untagged), Ubc9 and SENP1 constructs in the pCMV6-XL4 vector were from Origene Technologies (Rockville, MD).

Techniques: Expressing, Control, Recombinant

Journal: Journal of cell science

Article Title: SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability.

doi: 10.1242/jcs.036632

Figure Lengend Snippet: Fig. 3. SUMOylation regulates inactivation and recovery of Kv2.1 channels. (A)Representative current traces from HEK 293 cells expressing Kv2.1 alone (control) or with SUMO1-YFP (SUMO1), Ubc9 and/or SENP1 as indicated and normalized to peak current. (B)Inactivation time constants determined by fitting a single exponential decay function to the currents in panel A. (C)Recovery from inactivation following a 5-second depolarization to +30 mV in HEK 293 cells expressing Kv2.1 alone (black circles) or with SUMO1-YFP+Ubc9 (white triangles) or SUMO1-YFP+SENP1 (white circles). The Kv2.1+SUMO1-YFP curve was between the control and SENP1 curves and the Ubc9 curve (not shown). (D,E)The percent recovery to initial maximum current at 65.5 seconds and the recovery time constant determined by fitting the recovery curve to a single exponential function. (F)Voltage-dependence of steady-state inactivation of Kv2.1 expressed in HEK 293 cells either alone (black circles), with SUMO1-YFP (black triangles), with SUMO1-YFP+Ubc9 (white triangles) or with SUMO1-YFP+SENP1 (white circles). *P<0.05 and **P<0.01 compared with control; error bars, s.e.m.

Article Snippet: The human SUMO1 (untagged), Ubc9 and SENP1 constructs in the pCMV6-XL4 vector were from Origene Technologies (Rockville, MD).

Techniques: Expressing, Control

Journal: Journal of cell science

Article Title: SUMOylation regulates Kv2.1 and modulates pancreatic beta-cell excitability.

doi: 10.1242/jcs.036632

Figure Lengend Snippet: Fig. 4. SUMOylation regulates the excitability of INS1 832/13 β-cells. (A)Representative action potential traces induced by injection of a 10 pA current in INS1 832/13 cells expressing EGFP (control) or SUMO1-YFP (SUMO1), Ubc9 and/or SENP1 as indicated. (B-D)Action potential firing frequency, half-width and peak amplitudes for the groups shown in panel A. The half-width and peak amplitudes for the cells expressing SUMO1-YFP+Ubc9 were not analyzed because action potentials were rarely observed in this group. *P<0.05, **P<0.01 and ***P<0.001 compared with control; error bars, s.e.m.

Article Snippet: The human SUMO1 (untagged), Ubc9 and SENP1 constructs in the pCMV6-XL4 vector were from Origene Technologies (Rockville, MD).

Techniques: Injection, Expressing, Control