Journal: iScience

Article Title: Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

doi: 10.1016/j.isci.2021.103484

Figure Lengend Snippet: Senp5S competes with other Senps and promotes SUMOylation (A) Global SUMO3-ylation levels changed by expression of Senp5 isoforms. HEK293T cells were transfected with EGFP, EGFP-Senp3, EGFP-Senp5L, or EGFP-Senp5S along with HA-SUMO3 or HA-SUMO1, followed by immunoblotting with anti-HA (upper panel), anti-GFP (middle panel), or anti-α-tubulin (bottom panel). (B) Effect of Senp5L and Senp5S expression on SUMO3-ylation of Drp1. HEK293T expressing EGFP, EGFP-Senp5L, or EGFP-Senp5S along with Myc-Drp1 and HA-SUMO3 were immunoprecipitated (IP) with anti-HA antibody. Total cell lysates (input) or IP samples were analyzed by immunoblotting with anti-Myc (upper panel) and anti-HA (bottom panel). Immunoblot with an α-tubulin antibody as a loading control. SUMOylated-Drp1 is indicated by the perpendicular line. Heavy chain, immunoglobulin heavy chain of the HA antibody. (C) Domain structures of wild-type Drp1 (Drp1-WT) and a non-SUMOylatable Drp1 mutant (Drp1-4KR). The D-octadecapeptide sequence, comprising 18 amino acid residues (554–571) encompassing in the variable domain, is indicated. Drp1-4KR was created by substituting four SUMO-acceptor lysine residues ( red ) in the D-octadecapeptide with arginine residues. Oval and rectangle indicate GTPase activity domain and GTPase effector domain (GED), respectively. (D) Drp1 was SUMO3-ylated through the D-octadecapeptide sequence. HEK293 cells were co-transfected with HA-SUMO3, p14-Arf, Ubc9, and GFP, YFP-Drp1-WT or YFP-Drp1-4KR, then immunoprecipitated with anti-HA. Cell lysates (input) or IP samples were analyzed by immunoblotting with anti-GFP (top panel) or anti-HA (bottom panel). Immunoblot with an α-tubulin antibody as a loading control. SUMOylated-Drp1 is indicated by the perpendicular line.

Article Snippet: pcDNA3 HA-SUMO1 , , Addgene plasmid # 48966.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Sequencing, Activity Assay

Journal: iScience

Article Title: Drp1 SUMO/deSUMOylation by Senp5 isoforms influences ER tubulation and mitochondrial dynamics to regulate brain development

doi: 10.1016/j.isci.2021.103484

Figure Lengend Snippet:

Article Snippet: pcDNA3 HA-SUMO1 , , Addgene plasmid # 48966.

Techniques: Recombinant, Blocking Assay, Plasmid Preparation, Western Blot, Cell Culture, Mutagenesis, Software

Journal: Nature Communications

Article Title: Nur77 suppresses hepatocellular carcinoma via switching glucose metabolism toward gluconeogenesis through attenuating phosphoenolpyruvate carboxykinase sumoylation

doi: 10.1038/ncomms14420

Figure Lengend Snippet: ( a ) In DEN/CCl 4 -induced HCC samples (left) or normal livers (Ctrl) versus DEN/CCl 4 -induced HCC samples (right), endogenous PEPCK1 was first immunoprecipitated and then incubated with anti-PEPCK1 antibody or anti-SUMO1 antobody, respectively. Asterisk represents position of PEPCK1 protein; arrowheads indicate sumoylation bands. ( b ) The levels of PEPCK1 and its sumoylation in clinical carcinoma and paired para-carcinoma samples. Endogenous SUMO1 was immunoprecipitated and then incubated with anti-PEPCK1 antibody. ( c ) Top, HepG2 cell lysates with or without NEM (20 mM) was immunoprecipitated with anti-SUMO1 antibody and then incubated with anti-PEPCK1 antibody. Bottom, SUMO1 and Ubc9 were transfected into 293T cells, and Flag-SUMO1 was immunoprecipitated and then detected by western blot with anti-PEPCK1 antibody. ( d ) Sumoylation effect on endogenous PEPCK1 expression in different liver cancer cell lines that were transfected with SUMO1/Ubc9 (top) or treated with sumoylation inhibitor, anacardic acid (20 μM, 12 h) with or without SUMO1/Ubc9 transfection (middle and bottom). ( e ) Sumoylation attenuated PEPCK1 stability in SMMC-7721 cells that were transfected with different plasmids and then treated with CHX (100 μg ml −1 ) for different times (left). The amount of PEPCK1 protein was quantitated by software Image J (right). ( f ) Determination of the critical sites for PEPCK1 sumoylation in SMMC-7721 cells. The amount of PEPCK1 protein was normalized in each panel. ( g ) Lys124 site was important to stabilize PEPCK1 protein in SMMC-7721 cells with CHX (100 μg ml −1 ) treatment. ( h ) Sumoylation-induced PEPCK1 degradation via ubiquitination pathway in SMMC-7721 cells that were treated with MG132 (5 μM) or ALLM (20 μM) for 12 h. The expression level of PEPCK1 (left) or ubiquitination levels of PEPCK1, PEPCK1 K124R and PEPCK1 K471&473R (middle and right) were detected. NEM (20 mM) was added to cell lysates for repression of de-sumoylation in each sumoylation assay. Tubulin or GAPDH was used to indicate the amount of loading proteins. Data were represented as means±s.e.m. of at least three independent experiments. * P <0.05; ** P <0.01. The data were analysed using one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Anti-PCK1(cat. #D12F5; 1:2,000 dilution for western blot), anti-Nur77 (cat. #D63C5; 1:2,000 dilution for western blot), anti-SUMO1 (cat. # 4930S; 1:2,000 dilution for western blot), anti-acetylated lysine (cat. # 9681S; 1:2,000 dilution for western blot) and anti-snail (cat. #C15D3; 1:2,000 dilution for western blot) antibodies were from cell signalling technology.

Techniques: Immunoprecipitation, Incubation, Transfection, Western Blot, Expressing, Software

Journal: Nature Communications

Article Title: Nur77 suppresses hepatocellular carcinoma via switching glucose metabolism toward gluconeogenesis through attenuating phosphoenolpyruvate carboxykinase sumoylation

doi: 10.1038/ncomms14420

Figure Lengend Snippet: ( a ) p300-enhanced SUMO1/Ubc9-induced sumoylation in 293T cells. Different plasmids were transfected into cells, the sumoylation was indicated. ( b ) p300 inhibited endogenous SUMO1 and PEPCK1 levels in different liver cancer cell lines. p300 was transfected into cells and SUMO1 was immunoprecipetated and endogenous PEPCK1 sumoylation was indicated by its antibody. ( c ) p300-enhanced PEPCK1 sumoylation was dependent on its acetyltransferase activity. Different p300 inactive mutants were transfected into 293T cells as indicated. ( d ) p300 acetyltransferase activity was critical to enhance Ubc9 binding to PEPCK1 in 293T cells. ( e ) p300 could promote Ubc9, but not Ubc9 K65R , binding to PEPCK1 in 293T cells. ( f ) p300 interacted with Ubc9 K65R less than with Ubc9 in 293T cells (left), and p300 could acetylate Ubc9 but not Ubc9 K65R , detected with anti-acetylation antibody (Ac, right). ( g ) Ubc9 K65R had no effect on p300-enhanced PEPCK1 sumoylation in 293T cells. NEM (20 mM) was added to cell lysates for repression of de-sumoylation in each sumoylation assay.

Article Snippet: Anti-PCK1(cat. #D12F5; 1:2,000 dilution for western blot), anti-Nur77 (cat. #D63C5; 1:2,000 dilution for western blot), anti-SUMO1 (cat. # 4930S; 1:2,000 dilution for western blot), anti-acetylated lysine (cat. # 9681S; 1:2,000 dilution for western blot) and anti-snail (cat. #C15D3; 1:2,000 dilution for western blot) antibodies were from cell signalling technology.

Techniques: Transfection, Activity Assay, Binding Assay

Journal: Nature Communications

Article Title: Nur77 suppresses hepatocellular carcinoma via switching glucose metabolism toward gluconeogenesis through attenuating phosphoenolpyruvate carboxykinase sumoylation

doi: 10.1038/ncomms14420

Figure Lengend Snippet: ( a ) Nur77 abolished SUMO1/Ubc9-induced sumoylation (top), and p300-enhanced sumoylation (bottom) in different liver cancer cell lines. The amount of PEPCK1 sumoylation was quantitated by software Image J and presented under lanes of PEPCK1. ( b ) The role of Nur77 in elevating endogenous PEPCK1 expression levels in different liver cancer cell lines that were transfected with SUMO1 and Ubc9. ( c ) Nur77 elevated PEPCK1 protein expression in a time-dependent manner in HepG2 cells that were transfected with SUMO1 and Ubc9. ( d ) Nur77 blocked Ubc9 binding to PEPCK1 in 293T cells detected by co-IP assay. ( e ) Nur77 impaired p300 acetylation effect on PEPCK1 (left) or Ubc9 (right) detected with specific anti-acetylated lysine antibody (Ac) in 293T cells. ( f ) Images (left) and weight (right) of xenograft tumour in nude mice ( n =6). PEPCK1 or PEPCK1 K124R was stably overexpressed in Huh7 cells, and then Nur77 was further stably transfected into these cells. Cells were injected subcutaneously into the posterior flanks of nude mice. Scale bars, 1 cm. NEM (20 mM) was added to cell lysates for repression of de-sumoylation in each sumoylation assay. GAPDH was used to indicate the amount of loading proteins. Data were represented as means±s.e.m. of mice in the number indicated in the parenthesis. * P <0.05; *** P <0.001. The data were analysed using one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Anti-PCK1(cat. #D12F5; 1:2,000 dilution for western blot), anti-Nur77 (cat. #D63C5; 1:2,000 dilution for western blot), anti-SUMO1 (cat. # 4930S; 1:2,000 dilution for western blot), anti-acetylated lysine (cat. # 9681S; 1:2,000 dilution for western blot) and anti-snail (cat. #C15D3; 1:2,000 dilution for western blot) antibodies were from cell signalling technology.

Techniques: Software, Expressing, Transfection, Binding Assay, Co-Immunoprecipitation Assay, Stable Transfection, Injection

Journal: Scientific Reports

Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

doi: 10.1038/s41598-018-36312-4

Figure Lengend Snippet: sumo-1/2/3 and SUMO-1/2/3 levels increased as LMP1 levels increased in EBV-positive cell lines. ( a ) RNA was extracted from paired naïve lymphocytes, mitogen blasts, and EBV-transformed LCLs. cDNAs were generated, and real-time PCR was performed and relative sumo-1 , and sumo-2/3 levels (relative to gapdh ) determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate. Independent experiments were performed in triplicate. ( b – e ) RNA and protein were harvested from ten different EBV-positive cell lines. ( c ) cDNAs were generated, and real-time PCR was performed to quantitate relative LMP1, sumo-1 , and sumo-2/3 levels (relative to gapdh ). Results are shown as the mean for samples performed in triplicate from three independent experiments. Regression analysis of LMP1 and sumo-1 or sumo-2/3 levels was performed. ( d – f ) Slot immunoblots were performed to detect LMP1, SUMO-1, and SUMO-2/3 levels in ( d ) three EBV-transformed LCLs, ( e ) two KR4-HeLa fusion cell lines, and ( f ) three EBV-infected human breast cancer cell lines. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown.

Article Snippet: 24 hours post-transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, blocked with normal donkey serum, and stained with FLAG-specific (M2 Sigma) and SUMO-1-specific (FL-101; Santa Cruz Biotechnologies) antibodies overnight.

Techniques: Transformation Assay, Generated, Real-time Polymerase Chain Reaction, Standard Deviation, Western Blot, Infection

Journal: Scientific Reports

Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

doi: 10.1038/s41598-018-36312-4

Figure Lengend Snippet: LMP1 was necessary and sufficient to increase sumo-1/2/3 and SUMO-1/2/3 levels in vivo . ( a ) Real-time PCR was performed using cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells. The fold change in relative sumo- 1 and sumo- 2/3 expression (relative to gapdh ) was determined. Results are shown as the mean ± the standard deviation samples run in triplicate and independent experiments performed in triplicate. ( b – c ) 293 cells were serum-starved for 24 hours and then transfected with graduated amounts of an LMP1-expression vector or a control vector in serum-free media. ( b ) RNA was harvested, cDNA prepared, and real-time PCR performed. Relative LMP1, sumo-1 , and sumo-2/3 levels were determined (relative to gapdh ). Regression analysis of LMP1 and sumo-1 or sumo-2/3 levels was performed. Samples were run in triplicate and independent experiments performed in triplicate. ( c ) Slot blots were performed to detect SUMO-1, SUMO-2/3, and LMP1 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown.

Article Snippet: 24 hours post-transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, blocked with normal donkey serum, and stained with FLAG-specific (M2 Sigma) and SUMO-1-specific (FL-101; Santa Cruz Biotechnologies) antibodies overnight.

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Generated, Expressing, Standard Deviation, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

doi: 10.1038/s41598-018-36312-4

Figure Lengend Snippet: LMP1 CTAR1 and CTAR2 contributed to LMP1-mediated increase in sumo levels via the activation of NF-κB. ( a , b ) 293 cells were transfected with 1 μg of a LMP1-expression plasmid, select LMP1 mutant-expression plasmid, or a pcDNA3 control plasmid. ( a ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 and sumo-2/3 levels (relative to gapdh ), The fold change in sumo- 1 and sumo-2/3 levels (compared to control-expressing cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( b ) Slot blots were performed to detect SUMO-1 and SUMO-2/3 levels. Actin was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( c , d ) EBV-transformed LCLs were treated with Bay 11–7082 (1 μM), LY294002 (5 μM), or DMSO (the vehicle control; Control) for 24 hours. ( c ) RNA was harvested and cDNA was prepared. Real-time PCR was performed to quantitate relative sumo-1 , and sumo-2/3 expression (relative to gapdh ), and the fold change in sumo-1 and sumo-2/3 levels (relative to control-treated cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate. ( d ) Slot immunoblots and Western blots analyses were performed to detect SUMO-1 and SUMO-2/3 levels. GAPDH was used as a loading control. Representative blots for experiments performed in triplicate are shown. ( e ) cDNA generated from RNA harvested from BL41 EBV-negative, BL41 EBV WT, and BL41 EBV mut (P3HR1) cells that were treated with DMSO (the vehicle control; control) or Bay 11–7085 (1 μM) for 24 hours. Real-time PCR was performed was performed to quantitate relative sumo-1 , and sumo-2/3 levels (relative to gapdh ). The fold change in sumo-1 and sumo-2/3 levels (relative to control-treated BL41 EBV WT cells) was determined. Results are shown as the mean ± the standard deviation for samples performed in triplicate and independent experiments performed in triplicate.

Article Snippet: 24 hours post-transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, blocked with normal donkey serum, and stained with FLAG-specific (M2 Sigma) and SUMO-1-specific (FL-101; Santa Cruz Biotechnologies) antibodies overnight.

Techniques: Activation Assay, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Standard Deviation, Transformation Assay, Western Blot, Generated

Journal: Scientific Reports

Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

doi: 10.1038/s41598-018-36312-4

Figure Lengend Snippet: Increased sumo-1/2/3 levels were detected in LMP1-positive lymphoma tissues in an LMP1-dependent manner. ( a , b ) RNA was extracted from 42 biopsy tissues and cDNA was made. Real-time PCR was performed to quantitate gapdh , LMP1, and sumo-1 levels. RNA yields allowed sumo-2/3 levels to be tested in 17 of the 42 samples. ( a ) Relative LMP1, sumo-1 and sumo-2/3 levels (relative to gapdh ) were determined. The detection of LMP1 RNA levels determined is tissues were LMP1-negative (LMP1-neg) and LMP1-positive (LMP1-pos). Results are shown as the mean relative sumo-1 and sumo-2/3 levels for individual samples (shapes). Horizontal lines represent man sumo-1 and sumo-2/2 levels for the collective LMP1-neg and LMP1-pos samples. ( b ) Of the 19 LMP1-positive tissue samples, relative sumo- 1 levels were determined in all 19 samples but relative sumo-2/ 3 levels were determined in only 9 samples. Results are shown as the mean relative LMP1, sumo- 1, and sumo-2/ 3 leves of samples run in duplicate. Regression analysis was performed.

Article Snippet: 24 hours post-transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, blocked with normal donkey serum, and stained with FLAG-specific (M2 Sigma) and SUMO-1-specific (FL-101; Santa Cruz Biotechnologies) antibodies overnight.

Techniques: Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Epstein-Barr Virus Latent Membrane Protein-1 Induces the Expression of SUMO-1 and SUMO-2/3 in LMP1-positive Lymphomas and Cells

doi: 10.1038/s41598-018-36312-4

Figure Lengend Snippet: Increased SUMO levels were detected in LMP1-positive lymphoma tissues and cells. ( a ) FFPET biopsy tissues were sectioned onto glass slides. Following staining with rabbit anti-SUMO-1-specific and mouse anti-LMP1-specific primary antibodies and goat anti-mouse Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 594 secondary antibodies, coverslips were mounted using ProLong® Gold Antifade Reagent with DAPI. Immunofluorescence microscopy was performed at 20X magnification and images created using the Openlab software. ( b ) Serum-starved 293 cells were transfected with FLAG-LMP1-expression constructs. 24 hours post-transfection, cells were fixed, permeabilized, and stained with rabbit anti-SUMO-1-specific and mouse anti-FLAG-specific primary antibodies and goat anti-mouse Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 594 secondary antibodies. Coverslips were mounted using ProLong® Gold Antifade Reagent with DAPI. Confocal microscopy was performed at 60X magnification using the Nikon A1 laser confocal microscope. Images of LMP1-transfected cells next to non-LMP1-expressing cells were captured. Representative images are shown. ( c ) The corrected total cell fluorescence was determined for SUMO-1 levels in approximately 150 LMP1-negative and 150 LMP1-positive cells in lymphoma tissue samples and approximately 65 LMP1-negative and 65 LMP1-positive 293 cells. Results are shown as the mean ± the standard deviation.

Article Snippet: 24 hours post-transfection, cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, blocked with normal donkey serum, and stained with FLAG-specific (M2 Sigma) and SUMO-1-specific (FL-101; Santa Cruz Biotechnologies) antibodies overnight.

Techniques: Staining, Immunofluorescence, Microscopy, Software, Transfection, Expressing, Construct, Confocal Microscopy, Fluorescence, Standard Deviation

Journal: Cells

Article Title: Hepatitis B Core Protein Is Post-Translationally Modified through K29-Linked Ubiquitination

doi: 10.3390/cells9122547

Figure Lengend Snippet: HBc is not post-translationally modified by UBL proteins SUMO 1–3 and ISG15. Western blots of immunoprecipitated cell lysates with anti-FLAG or anti-HA magnetic beads after transient co-transfection of Huh7 cells with HBc and the UBL proteins SUMO-1 ( a ), SUMO-2 ( b ), SUMO-3 ( c ), and ISG15 ( d ). The cells were harvested 48 h post-transfection and precleared cell lysates (1 mg of proteins, WB: Input 10 µg of total protein/lane) were immunoprecipitated with anti-FLAG, or anti-HA magnetic beads. HBc was detected using rabbit polyclonal and tags antibodies. The position of IgG light chains is indicated by *.

Article Snippet: The MYC-DDK-tagged SUMO-1 (RC200633, SUMO-1) and MYC-DDK-tagged ISG15 (RC2012353, ISG15) plasmids were purchased from OriGene (Rockville, MD, USA).

Techniques: Modification, Western Blot, Immunoprecipitation, Magnetic Beads, Cotransfection, Transfection