Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 1 TRIM32 expression is decreased in failing human hearts and hypertrophic mouse hearts (A and B) Representative Western blots (A) and quantitative results (B) of ANP, β-MHC and TRIM32 protein expression in normal donor hearts and in human hearts with DCM (n = 8 hearts per experimental group; *P < 0.05 compared with donor hearts). (C and D) Representative Western blots (C) and quantitative results (D) of ANP, β-MHC and TRIM32 protein expression in a hypertrophic mouse heart induced by AB for the times indicated (W, weeks) (n = 6 mice per experimental group; *P < 0.05 compared with sham). (E and F) Representative Western blots (E) and quantitative results (F) of ANP, β-MHC and TRIM32 protein expression in cultured NRCMs treated with AngII for 24 or 48 h (n = 4 samples per experimental group; four independent experiments; *P < 0.05 compared with PBS). Results are means +−S.D., and statistical analyses were determined using an unpaired Student’s t test. Molecular masses are indicated in kDa in the Western blot panels.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Expressing, Western Blot, Cell Culture

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 2 TRIM32 attenuates AngII-induced cardiomyocyte hypertrophy in vitro (A) The protein expression level of TRIM32 in NRCMs after infection with AdshRNA, AdshTRIM32, AdGFP or AdTRIM32 was determined by Western blotting (n = 4 samples per experimental group). Left: representative blots. Right: quantitative results. (B) Representative images of NRCMs infected with AdshRNA, AdshTRIM32, AdGFP or AdTRIM32 and treated with AngII (1 μmol/l) or PBS for 48 h (n = 4 samples per experimental group; blue, nucleus; green, α-actinin; scale bar, 20 μm). (C) Quantitative results of the cell surface area of NRCMs infected with AdshTRIM32 or AdshRNA subjected to AngII (1 μmol/l) or PBS for 48 h (n⩾50 cells per experimental group; *P < 0.05 compared with AdshRNA/PBS; #P < 0.05 compared with AdshRNA/AngII). (D) Quantitative results of the cell surface area of NRCMs infected with AdTRIM32 or AdGFP subjected to AngII (1 μmol/l) or PBS for 48 h (n⩾50 cells per experimental group; *P < 0.05 compared with AdGFP/PBS; #P < 0.05 compared with AdGFP/AngII). (E) Relative mRNA levels of ANP, BNP and β-MHC in AdshTRIM32-infected NRCMs

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: In Vitro, Expressing, Infection, Western Blot

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 3 TRIM32 overexpression attenuates pressure overload-induced cardiac hypertrophy and heart failure (A) Schematic diagram of the construction of TG mice with a full-length murine Trim32 cDNA under the control of the α-MHC promoter. (B and C) Representative Western blots (B) and quantitative results (C) of TRIM32 protein expression in heart tissue from four TG lines and MCT mice (n = 3 mice per experimental group; three independent experiments).

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Over Expression, Control, Western Blot, Expressing

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 4 Generation of global TRIM32-deficient mice (A) One sgRNA targeted a region downstream of the 5′ end of exon 2 in the Trim32 mouse gene. (B) Representative results of the T7E1 assay from pups subjected to microinjection. Sizes are indicated in bp. (C) Representative results of the DNA sequencing from mice with frameshift mutations. (D) Agarose gel photograph illustrating genotyping results of PCR products from WT (+/+), heterozygous (+/−) and TRIM32-KO (−/−) mice. Sizes are indicated in bp. (E) Representative Western blots of TRIM32 expression in heart tissues from global TRIM32-KO mice and their littermate controls (n = 4 mice per experimental group). Molecular masses are indicated in kDa.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Microinjection, DNA Sequencing, Agarose Gel Electrophoresis, Western Blot, Expressing

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 5 TRIM32 deficiency aggravates pressure overload-induced cardiac hypertrophy and heart failure (A) Statistical results for HW/BW and HW/TL in WT mice and TRIM32-KO mice at 4 weeks after sham or AB (n = 8–10 mice per experimental group). (B) Representative images of gross morphology, H&E staining, WGA staining and PSR staining of hearts from the indicated groups (n = 5 mice per experimental group; scale bar, 20 μm for lower H&E staining,

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Staining

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 6 TRIM32 inhibits activation of Akt-dependent signalling pathways upon hypertrophic stresses

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Activation Assay

Journal: Clinical Science

Article Title: Tripartite motif 32 prevents pathological cardiac hypertrophy

doi: 10.1042/cs20150619

Figure Lengend Snippet: Figure 7 Blockage of Akt-dependent signalling pathways rescues pressure overload-induced cardiac abnormalities in TRIM32-KO mice (A and B) Representative Western blots (A) and quantitative results (B) of total (T-) and phospho- (P-) Akt-dependent signalling pathway protein levels in the hearts from LY294002- or DMSO-treated TRIM32-KO mice 4 weeks after AB surgery (n = 4 mice per experiment group; GAPDH was used as a loading control). Molecular masses are indicated in kDa in (A). (C) Statistical results for HW/BW and HW/TL in the indicated groups (n = 8–10 mice per experimental group). (D) Representative images of gross morphology, H&E staining and PSR staining from hearts in the indicated groups at 4 weeks after AB (n = 5 mice per experimental group; scale bar, 20 μm for lower H&E staining and PSR staining). (E) Statistical results for the cardiomyocyte cross-sectional area in the indicated groups (n⩾100 cells per experimental group). (F) Quantification of left ventricular collagen volume in the indicated groups (n⩾25 fields per experimental group). (G) Measurements of LVESD, LVEDD and FS in the indicated groups (n = 5 mice per experimental group). *P < 0.05 compared with KO/AB/DMSO; n.s. indicates no significant difference. Results are means +−S.D., and statistical analyses were performed using an unpaired Student’s t test or one-way ANOVA.

Article Snippet: The antibody against TRIM32-(28–984) was from ProSci.

Techniques: Western Blot, Control, Staining

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 2. Trim32 is recruited to spindle poles during mitosis through Cdk1/cyclin B phosphorylation signaling. A,representative images of localization of Trim32 to spindle poles during mitosis but not in interphase in SH-SY5Y, TGW, and SK-N-DZ cells. Centrosome marker pericentrin, green; Trim32, red; DAPI (DNA), blue. Arrows, spindle poles. B and C, representative images of Trim32 localization to spindle poles during mitosis in TGW and SK-N-DZ cells upon exposure to 5 mmol/L RO-3306 for 3 hours. DMSO (0.1%)-treated cells were used as a control. Centrosome (centriole) marker centrin-2, is green; Trim32, red; DAPI (DNA), blue. Arrows, spindle poles. D, immunoblot of cyclin B expression in SK-N-DZ cells transfected with control siRNA or Cyclin B siRNA. Immunoblot of b-actin served as a loading control. E, representative images of Trim32 localization to spindle poles during mitosis in SK-N-DZ cells transfected with control siRNA or Cyclin B siRNA. Centrin-2, green; Trim32, red; DAPI (DNA), blue. Arrows, spindle poles. F, percentage of cells with Trim32 localization to spindle poles in SK-N-DZ cells transfected with control siRNA or Cyclin B siRNA. Error bars, SEM from three experiments. Localization statuses of Trim32 at spindle poles were categorized into three types (intense, weak, or no localization). G, representative images of Flag-Trim32 or Flag-Trim32/3A localization to spindle poles during mitosis in SK-N-DZ cells transfected with Flag-Trim32 or Flag-trim32/3A expression vector. Pericentrin, green; Flag, red; DAPI (DNA), blue. Arrows, spindle poles. Flag-Trim32 localizes to spindle poles, but Flag-Trim32/3A does not. Scale bars, 10 mm.

Article Snippet: The primary antibodies used were anti-GSK-3b antibody (sc-7291; Santa Cruz Biotechnology), anti-cyclin B antibody (sc-245; Santa Cruz Biotechnology), anti-b-actin antibody (#612656; BD Biosciences), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Trim32 antibody (sc-53993; Santa Cruz Biotechnology), anti-Trim32 antibody (sc-9011; Santa Cruz Biotechnology), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (sc-791; Santa Cruz Biotechnology), anti-GST antibody (PM013 and MBL), and anti-g-tubulin monoclonal antibody (clone GTU-88; Sigma).

Techniques: Phospho-proteomics, Marker, Control, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 3. MYCN is a physiologic substrate of Trim32 ubiquitin ligase. A, one milligram of cell lysate from TGW cells was immunoprecipitated with an anti-MYCN antibody. As a control, immunoprecipitates with preimmune mouse immunoglobulin-G (IgG) were used. The immunoprecipitates were then subjected to immunoblotting using an anti-Trim32 antibody (left). Conversely, 1 mg of cell lysate was immunoprecipitated with an anti-Trim32 antibody, and the immunoprecipitates were subjected to immunoblotting using an anti-MYCN antibody. As a control, immunoprecipitates with preimmune mouse IgG were used (right). B, Trim32 directly ubiquitinylates MYCN in vitro. Trim32 immunoprecipitates from TGW cells were mixed with GST or recombinant GST-MYCN with or without ubiquitin, and an in vitro ubiquitylation assay was performed. C, immunoblot of Trim32 and MYCN expression in TGW cells transfected with control siRNA or Trim32 siRNA. Immunoblot of g-tubulin served as a loading control. D, representative images of MYCN localization to spindle poles during mitosis in TGW cells transfected with control siRNA or Trim32 siRNA. NuMA, green; MYCN, red; DAPI (DNA), blue. Arrows, spindle poles. Scale bars, 10 mm. E, percentage of cells with MYCN-localized spindle poles in TGW cells transfected with control siRNA or Trim32 siRNA. Error bars, SEM from three experiments. Y-axis, percentage of mitotic cells with MYCN-localized spindle poles.

Article Snippet: The primary antibodies used were anti-GSK-3b antibody (sc-7291; Santa Cruz Biotechnology), anti-cyclin B antibody (sc-245; Santa Cruz Biotechnology), anti-b-actin antibody (#612656; BD Biosciences), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Trim32 antibody (sc-53993; Santa Cruz Biotechnology), anti-Trim32 antibody (sc-9011; Santa Cruz Biotechnology), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (sc-791; Santa Cruz Biotechnology), anti-GST antibody (PM013 and MBL), and anti-g-tubulin monoclonal antibody (clone GTU-88; Sigma).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Control, Western Blot, In Vitro, Recombinant, Ubiquitin Assay, Expressing, Transfection

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 4. Trim32 may be an inducer of ACD against MYCN function. TGW cells show approximately 10% NuMA cortex based–asymmetric cell division (N-ACD). A, TGW cells were transfected with Trim32 siRNA, and we then counted the percentage of NuMA-ACD cells with or without Trim32 knockdown. A, percentage of TGW cells with NuMA-ACD transfected with control or Trim32 siRNA. Error bars, SEM from three experiments; P ¼ 0.0163. B, representative images of NuMA-ACD in TGW with control siRNA or Trim32 siRNA. Cell cortex marker NuMA, green; g-tubulin, red; DAPI (DNA), blue. Arrows, NuMA-cortex. Percentage of TGW cells with NuMA-ACD was significantly reduced by knockdown of Trim 32.

Article Snippet: The primary antibodies used were anti-GSK-3b antibody (sc-7291; Santa Cruz Biotechnology), anti-cyclin B antibody (sc-245; Santa Cruz Biotechnology), anti-b-actin antibody (#612656; BD Biosciences), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Trim32 antibody (sc-53993; Santa Cruz Biotechnology), anti-Trim32 antibody (sc-9011; Santa Cruz Biotechnology), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (sc-791; Santa Cruz Biotechnology), anti-GST antibody (PM013 and MBL), and anti-g-tubulin monoclonal antibody (clone GTU-88; Sigma).

Techniques: Transfection, Knockdown, Control, Marker

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 5. Trim32/3A has ubiquitinylation activity for MYCN, but induces aggresome. A, representative images of Flag- and MYCN-stained SK-N-DZ cells transfected with the Flag-Trim32 or Flag-Trim32/3A expression vector. Flag, green; MYCN, red; DAPI (DNA), blue. Arrowheads, untransfected cells. While MYCN signals were detected in untransfected cells, MYCN signals were not detected in Flag-Trim32- and Flag-Trim32/3A-transfected cells. Percentage of MYCN-negative/Flag-positive cells is shown on the right side. Error bars, SEM from three experiments. B, representative images of Flag- and MYCN-stained SK-N-DZ cells transfected with the Flag-trim32 or Flag-Trim32/3A expression vector. Flag, green; MYCN, red; DAPI (DNA), blue. Arrows, aggresomes in Flag-Trim32-transfected cells. Arrowheads, aggresomes in Flag-Trim32/3A–transfected cells. Percentage of aggresome-positive cells is shown in right. Error bars, SEM from three experiments; P ¼ 0.0005.

Article Snippet: The primary antibodies used were anti-GSK-3b antibody (sc-7291; Santa Cruz Biotechnology), anti-cyclin B antibody (sc-245; Santa Cruz Biotechnology), anti-b-actin antibody (#612656; BD Biosciences), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Trim32 antibody (sc-53993; Santa Cruz Biotechnology), anti-Trim32 antibody (sc-9011; Santa Cruz Biotechnology), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (sc-791; Santa Cruz Biotechnology), anti-GST antibody (PM013 and MBL), and anti-g-tubulin monoclonal antibody (clone GTU-88; Sigma).

Techniques: Activity Assay, Staining, Transfection, Expressing, Plasmid Preparation

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 6. Trim32 suppresses sphere formation in human neuroblastoma cells. A, representative images of sphere formation in Flag (control)-transfected, Flag-Trim32–transfected, and Flag-Trim32/3A–transfected SK-N-DZ after 7 days. Scale bars, 100 mm. B, numbers of spheres of 100 mm in each transfectant. Error bars, SEM from triplicate experiments. The numbers of spheres of 100 mm were lower in Flag-Trim32–transfected cells (P ¼ 0.0009) and in Flag-Trim32/32A–transfected cells (P ¼ 0.026) than in Flag (control)-transfected ones.

Article Snippet: The primary antibodies used were anti-GSK-3b antibody (sc-7291; Santa Cruz Biotechnology), anti-cyclin B antibody (sc-245; Santa Cruz Biotechnology), anti-b-actin antibody (#612656; BD Biosciences), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Trim32 antibody (sc-53993; Santa Cruz Biotechnology), anti-Trim32 antibody (sc-9011; Santa Cruz Biotechnology), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (sc-791; Santa Cruz Biotechnology), anti-GST antibody (PM013 and MBL), and anti-g-tubulin monoclonal antibody (clone GTU-88; Sigma).

Techniques: Control, Transfection

Journal: Cancer Research

Article Title: Trim32 Facilitates Degradation of MYCN on Spindle Poles and Induces Asymmetric Cell Division in Human Neuroblastoma Cells

doi: 10.1158/0008-5472.can-14-0169

Figure Lengend Snippet: Figure 7. Trim32 induces ACD in human neuroblastoma cells. A, immunoblot of Trim32, MYCN, and CD133 (a putative neural stem cell marker) expression in SK-N-DZ cells transfected with the Trim32 or Trim32/3A expression vector. Immunoblot of g-tubulin served as a loading control. B, representative images of ACD during anaphase in SK-N-DZ cells transfected with the Trim32 or Trim32/3A expression vector. Cell cortex marker NuMA, green; Trim32, red; DAPI (DNA), blue. SCD, symmetric cell division; NuMA-ACD, NuMA-cortex–based ACD; complete ACD, cell division in which NuMA and Trim32 were distributed at opposite ends of a dividing cell; and sympatric ACD, cell division in which NuMA and Trim32 were distributed at the same end of a dividing cell. Arrows, NuMA cortex; arrowheads, Trim32 distribution; red arrowheads, spindle poles. Scale bars, 10 mm. C, percentage of ACD in SK-N-DZ cells transfected with the Trim32 or Trim32/3A expression vector. Error bars, SEM from three experiments. Complete ACD is more frequent in Trim32- transfected cells than in control vector–transfected cells (P ¼ 0.0046) or in Trim32/3A–transfected ones (P ¼ 0.0064). D, a schematic model of Trim32- mediated ACD in MYCN-amplified human neuroblastoma cells.

Article Snippet: The primary antibodies used were anti-GSK-3b antibody (sc-7291; Santa Cruz Biotechnology), anti-cyclin B antibody (sc-245; Santa Cruz Biotechnology), anti-b-actin antibody (#612656; BD Biosciences), anti-Trim32 antibody (H00022954-M09; Abnova), anti-Trim32 antibody (sc-53993; Santa Cruz Biotechnology), anti-Trim32 antibody (sc-9011; Santa Cruz Biotechnology), anti-MYCN antibody (sc-53993; Santa Cruz Biotechnology), anti-MYCN antibody (sc-791; Santa Cruz Biotechnology), anti-GST antibody (PM013 and MBL), and anti-g-tubulin monoclonal antibody (clone GTU-88; Sigma).

Techniques: Western Blot, Marker, Expressing, Transfection, Plasmid Preparation, Control

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Western blot analysis of cell extracts from HEK293 FlpIn cells with tetracycline inducible expression of EGFP-TRIM32 WT , the BBS11 associated mutant EGFP-TRIM32 P130S , or the LGMD2H causing mutant EGFP-TRIM32 D487N shows auto-ubiquitylation activity of TRIM32 WT and TRIM32 P130S , but not the LGMD2H mutant. (B) Sequence of the TRIM32 peptides identified by MS to display PTMs on lysine residues that differed between the TRIM32 proteins with auto-ubiquitylation activity (TRIM32 WT and TRIM32 P130S ) and the LGMD2H disease variant lacking auto-ubiquitylation activity (TRIM32 D487N ). (C) Schematic of TRIM32 domain organization. The D487N mutation in the NHL repeats that is associated with LGMD2H and the P130S mutation in the B-box causing BBS11 are indicated above. The three lysine residues that are focused in this paper are indicated in red, while the PTMs of these residues identified by MS are indicated above. Ub: Ubiquitylation. Ac: Acetylation (D) Western Blot analysis of HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , the double mutants EGFP-TRIM32 K247R/K401R , EGFP-TRIM32 K50R/K247R , or EGFP-TRIM32 K247R/K401R , or the triple mutant EGFP-TRIM32 K50R/K247R/K401R . The last lane represents co-transfection of EGFP-TRIM32 WT and mCherry-USP2 expression vectors. The bands representing EGFP-TRIM32, auto-ubiquitylated EGFP-TRIM32, and cleaved EGFP-TRIM32 are indicated to the right. PCNA represents the loading control. (E) Western blot analysis of a similar experiment as in (D), except that the myoblast C2C12 cell line is used instead of the HEK293 FlpIn TRIM32 KO cell line, and the antibody used is an anti-TRIM32 antibody instead of an anti-GFP antibody.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Western Blot, Expressing, Mutagenesis, Activity Assay, Sequencing, Variant Assay, Transfection, Cotransfection, Control

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Schematic of the domain organization of TRIM32 isoform 1 and TRIM32 isoform 3. (B) Western Blot analysis of HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 K50R/K247R/K401R , EGFP-TRIM32 K50Q/K247R/K401R , TRIM32 K50R/K247Q/K401R or EGFP-TRIM32 K50R/K247R/K401Q . The bands representing EGFP-TRIM32 and cleaved EGFP-TRIM32 are indicated to the right. Ponceau staining of the membrane represents the loading control. (C) Confocal images of HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 K50R/K247R/K401R , EGFP-TRIM32 K50Q/K247R/K401R , TRIM32 K50R/K247Q/K401R or EGFP-TRIM32 K50R/K247R/K401Q , and immunostained with anti-GM130 antibodies, a Golgi marker. The cell nuclei are visualized by DAPI staining. Scale bars: 10 μM.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Western Blot, Transfection, Expressing, Staining, Membrane, Control, Marker

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Confocal images of mCherry-EYFP-TRIM32 WT , mCherry-EYFP-TRIM32 K247R/K401R , mCherry-EYFP-TRIM32 K247Q , mCherry-EYFP-TRIM32 K50R/K247Q/K401R or mCherry-EYFP-TRIM32 K50R/K247R/K401R transiently expressed in HEK293 FlpIn TRIM32 KO cells and exposed to normal or starved conditions. Scale bars: 10 μM. (B) The graphs represent the number of cells with RedOnly dots in the mCherry-EYFP-TRIM32 transfected cells shown with representative images in A. The graphs represent the average of three independent experiments with s.d. (n>20 cells). **P< 0.005; ***P<0.0005 (Student’s t -test).

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Transfection

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: (A) Schematic of TRIM32 domain organization with the three regulatory Lysine residues identified in this work indicated above, and the sequence of the mapped PEST sequence indicated below. MutPEST and ΔPEST display the modifications introduced to the predicted PEST sequence. (B) Western Blot analysis of cell extracts from HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 mutPEST , EGFP-TRIM32 ΔPEST , EGFP-TRIM32 K50R/K247R/K401R (3KR), TRIM32 K50R/K247R/K401R/ΔPEST (3KR ΔPEST) or EGFP-TRIM32 K247Q . The bands representing EGFP-TRIM32 and cleaved EGFP-TRIM32 are indicated to the right. Tubulin represents the loading control. (C) Western Blot analysis of cell extracts from HEK293 FlpIn TRIM32 KO cells transiently transfected with expression plasmids for EGFP-TRIM32 WT , EGFP-TRIM32 WT and mCherry-USP2, EGFP-TRIM32 ΔPEST , EGFP-TRIM32 K50R/K247R/K401R (3KR), TRIM32 K50R/K247R/K401R/ΔPEST (3KR ΔPEST) or EGFP-TRIM32 K50R/247Q/K401R or EGFP-TRIM32 D487N . The bands representing ubiquitylated EGFP-TRIM32, EGFP-TRIM32 and cleaved EGFP-TRIM32 are indicated to the right. Actin represents the loading control.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques: Sequencing, Western Blot, Transfection, Expressing, Control

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques:

Journal: PLoS ONE

Article Title: Generation of the short TRIM32 isoform is regulated by Lys 247 acetylation and a PEST sequence

doi: 10.1371/journal.pone.0251279

Figure Lengend Snippet: Oligonucleotides used in this study.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody for TRIM32 (Proteintech, 10326-1-AP); rabbit polyclonal anti-GFP (Abcam, ab290); rabbit polyclonal anti-Actin (Sigma, A2066); mouse monoclonal anti-PCNA (DAKO, M0879); rabbit monoclonal anti-GM130 (Abcam, #52649).

Techniques:

Journal: European journal of pharmacology

Article Title: The enhancing effect of 5-HT on phasic contractions of human isolated distal ureter and the mechanisms mediating these effects.

doi: 10.1016/j.ejphar.2023.175715

Figure Lengend Snippet: Fig. 4. The blocking effect of 5-HT2A selective an tagonists on 5-HT stimulatory action. 5-HT concentration-response curves for frequency (A) and baseline tone (B) were established in the presence or absence of volinanserin (1, 10, 100 nM), a 5-HT2A selective antagonist. Volinanserin shifted the 5-HT centration-response curve of baseline tension right ward, but did not have significant blocking effects on the centration-response curve of frequency. For ten sion change, the Schild plot for volinanserin revealed a pA2 value of 8.18 with a slope of 0.39 ± 0.03 (B). Contractions are expressed as a percentage of the calculated maximal effect. Each curve was created using the average data of five ureter strips.

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4 ◦C with primary antibodies against the following proteins: 5-HT2A receptor (1:500, A20538; ABclonal, China), 5-HT2C receptor (1:500, DF3501; Affinity, China), and GAPDH (1:1000, AC001; ABclonal, China).

Techniques: Blocking Assay, Concentration Assay

Journal: European journal of pharmacology

Article Title: The enhancing effect of 5-HT on phasic contractions of human isolated distal ureter and the mechanisms mediating these effects.

doi: 10.1016/j.ejphar.2023.175715

Figure Lengend Snippet: Fig. 3. The relative mRNA expression level of 5-HT receptors in human distal ureter. Total RNA was extracted from human distal ureter tissue, and RT- qPCR was performed. The primer sets for the 13 5- HT receptors are indicated in Supplementary Table 1. Because the expression level of the 5-HT receptor was much lower than that of beta-actin, the mRNA expression of each receptor was expressed as a relative level to that of 5-HT2A, which showed the highest expression in all receptors. Sum mary data are the average from six experiments.

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4 ◦C with primary antibodies against the following proteins: 5-HT2A receptor (1:500, A20538; ABclonal, China), 5-HT2C receptor (1:500, DF3501; Affinity, China), and GAPDH (1:1000, AC001; ABclonal, China).

Techniques: Expressing, Quantitative RT-PCR

Journal: European journal of pharmacology

Article Title: The enhancing effect of 5-HT on phasic contractions of human isolated distal ureter and the mechanisms mediating these effects.

doi: 10.1016/j.ejphar.2023.175715

Figure Lengend Snippet: Fig. 6. 5-HT2C selective agonist increased the frequency of ureter phasic con tractions in a concentration-dependent manner. Concentration-response curves to 5-HT, vabicaserin (5-HT2C selective agonist), and 25CN–NBOH (5-HT2A se lective agonist) were established. Both vabicaserin and 25CN–NBOH did not induce baseline increase, and only frequency changes were analyzed. The maximal response induced by vabicaserin and 25CN–NBOH was 35% and 12% of 5-HT, respectively. Each curve was prepared using the average data of five ureter strips.

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4 ◦C with primary antibodies against the following proteins: 5-HT2A receptor (1:500, A20538; ABclonal, China), 5-HT2C receptor (1:500, DF3501; Affinity, China), and GAPDH (1:1000, AC001; ABclonal, China).

Techniques: Concentration Assay

Journal: European journal of pharmacology

Article Title: The enhancing effect of 5-HT on phasic contractions of human isolated distal ureter and the mechanisms mediating these effects.

doi: 10.1016/j.ejphar.2023.175715

Figure Lengend Snippet: Fig. 9. 5-HT2A/2C receptor protein expression in human distal ureter. (A) Western blot experiments show 5-HT2A (left) and 5-HT2C (right) expression in ureter tissue from the mucosal or intact layer. Bands for 5-HT2A at the expected weight of 55 kDa and bands for 5-HT2C at expected weight of 53 kDa. GAPDH was used as the control. (B) Immunofluores cence labeling showed that 5-HT2A (left) or 5-HT2C (right) receptors are expressed on nerve terminals, interstitial cells, and on the urothelium of human ureter.

Article Snippet: After blocking with 5% skim milk, the membranes were incubated overnight at 4 ◦C with primary antibodies against the following proteins: 5-HT2A receptor (1:500, A20538; ABclonal, China), 5-HT2C receptor (1:500, DF3501; Affinity, China), and GAPDH (1:1000, AC001; ABclonal, China).

Techniques: Expressing, Western Blot, Control, Labeling

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: TRIM32 restricts SARS-CoV-2 replication in a RING-dependent but IFN-independent manner ( A ) Validation of CRISPR TRIM32- KO Caco-2 cells. TRIM32 protein abundance in TRIM32 -KO Caco-2 single cell clones (cl-1 and cl-2) and WT control cells, determined in the whole cell lysates (WCLs) by immunoblot (IB) analysis with anti-TRIM32 and anti-Actin (loading control). Asterisk indicates nonspecific band. ( B ) Validation of CRISPR TRIM32- KO Caco-2 cells (cl-1 and cl-2) by assessing TRIM32 transcripts by RT-qPCR. Values indicate relative mRNA (fold), normalized to cellular HPRT1 . ( C ) Virus replication kinetics over a 72-h period in TRIM32 -KO and WT Caco-2 cells that were either mock-treated or infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02), determined by measuring RFU for EGFP. Values were auto-scaled to the average values for mock-infected cells at 0 h, set to 200. ( D and E ) RT-qPCR analysis of SARS-CoV-2 g. Nsp3 (D) and sg. N&Orf9b (E) transcripts in TRIM32 -KO and WT Caco-2 cells that were either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h. ( F ) SARS-CoV-2 spike (S) and nucleocapsid (N) protein abundances in TRIM32 -KO and WT Caco-2 cells that were mock-treated (−) or infected as in (D), determined in the WCLs by IB with anti-S and anti-N. ( G ) TRIM32 protein expression in stable reconstituted TRIM32 -KO Caco-2 cells that inducibly (via doxycycline (DOX; 2 ug/mL) treatment for 24 h) expressed either empty vector or FLAG-tagged TRIM32 WT or ΔRING, determined in the WCLs by IB with anti-TRIM32, anti-FLAG, and anti-Actin (loading control). WT Caco-2 cells expressing empty vector served as control. Asterisk indicates nonspecific band. ( H ) Virus replication kinetics over a 72-h period in TRIM32 -KO Caco-2 cells that were reconstituted as in (G) and then either mock-treated or infected with EGFP-rSARS-CoV-2 (strain K49; MOI 0.02), determined by measuring RFU for EGFP. Values were auto-scaled as in (C). WT Caco-2 cells expressing empty vector served as control. ( I ) SARS-CoV-2 g. Nsp3 transcripts in TRIM32 -KO Caco-2 cells reconstituted as indicated and either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 0.02) for 48 h, determined by RT-qPCR. WT Caco-2 cells expressing empty vector served as control. ( J and K ) SARS-CoV-2 g. Nsp3 and sg. N&orf9b transcripts in stable Caco-2 cells that inducibly (via DOX (2 ug/mL) treatment) expressed empty vector (control) or TRIM32 WT and were infected with SARS-CoV-2 (strain WA1; MOI 0.05) for 48 h in the absence or presence of anti-IFNAR2 antibody (2 µg/mL), determined by RT-qPCR. Data are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates (B—E, H—K)). **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). ns, not significant. See also Figure S2.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Biomarker Discovery, CRISPR, Quantitative Proteomics, Clone Assay, Control, Western Blot, Quantitative RT-PCR, Virus, Infection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: TRIM32 binds to and SUMOylates SARS-CoV-2 NSP14 ( A ) Endogenous TRIM32 binding to NSP14 in Caco-2 cells that were either mock-treated (−) or infected with SARS-CoV-2 (strain WA1; MOI 1) for 48 h, determined by IP with anti-TRIM32 and IB with anti-NSP14. ( B ) Schematic representation of TRIM32’s domain organization as well as the truncation constructs used in the interaction-mapping experiments. RING, really interesting new gene; BB, B-box; CC, coiled-coil; NHL, NCL-1, HT2A, Lin-41 domain; mutSIM, a mutant of TRIM32 in which the SIM motif located in the NHL domain is mutated. Mutated residues are shown in red. Numbers indicate amino acids. ( C ) Binding of FLAG-NSP14 (SARS-CoV-2) to the indicated V5-tagged TRIM32 truncation proteins in transiently transfected HEK293T cells, assessed by PD with anti-V5 (PD: V5), followed by IB with anti-FLAG. WCLs were probed with anti-FLAG and anti-Actin (loading control). ( D ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) in HEK293T cells that were co-transfected for 40 h with V5-tagged TRIM32 WT or ΔRING, determined by PD: FLAG and IB with anti-pan-SUMO. WCLs were probed by IB with anti-V5 and anti-Actin (loading control). ( E ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) in transiently transfected HEK293T cells that co-expressed either empty vector or V5-tagged TRIM32 WT or ΔRING, along with UBC9-HA and Myc-tagged SUMO1, SUMO2 or SUMO3, determined by PD: FLAG and IB with anti-Myc and anti-FLAG. Expression of co-transfected proteins was determined in the WCLs using the indicated antibodies. ( F ) SUMOylation of NSP14 in stable Caco-2 cells that were infected for 32 h with SARS-CoV-2 (strain WA1; MOI 2) and then treated for 12 h with DOX (2 µg/mL) to inducibly express FLAG-tagged TRIM32, assessed by denaturing IP with anti-SUMO2/3 and IB with anti-NSP14. WCLs were probed by IB with the indicated antibodies. Hc, antibody heavy chain. Asterisk indicates nonspecific band. ( G ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) in HEK293T cells that were transfected for 40 h with either empty vector or V5-tagged TRIM32 WT or mutants (ΔRING or mutSIM) together with Myc-tagged SUMO2 and HA-tagged UBC9, determined by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with the indicated antibodies. Data (A and C—G) are representative of at least two independent experiments. See also Figure S3.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Binding Assay, Infection, Construct, Mutagenesis, Transfection, Control, Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: The NSP14 ExoN domain is SUMOylated at K9 and K200 ( A ) Schematic representation of the approach to identify the SUMOylation sites in NSP14 of SARS-CoV-2 by mass spectrometry (MS) analysis. HEK293T cells were transiently transfected with FLAG-tagged NSP14, V5-tagged TRIM32, HA-tagged UBC9, and Myc-tagged SUMO2 T91R mutant. 40 h later, NSP14 was affinity-purified by PD: FLAG, followed by SDS-PAGE and Coomassie blue staining. Unmodified NSP14 (arrowhead) and modified NSP14 versions are indicated (center). NSP14 protein was subjected to digestion using trypsin, and peptides modified by SUMO2 T91R Lys-Gly-Gly (KGG) signatures were analyzed using LC-MS/MS. Asterisk indicates antibody heavy chain. Parts of this figure were created using Biorender.com. ( B ) Top: Schematic representation of SARS-CoV-2 NSP14 domain organization with the SUMOylated lysines (Ks) identified by MS analysis indicated. ExoN, exonuclease domain; N7-MTase, N7-methyltransferase. Bottom: Mass spectra of the tryptic peptides of affinity-purified FLAG-tagged NSP14 (SARS-CoV-2) that identified SUMOylation at K9 and K200, with designated b-and y-ions. Ace denotes acetylated N-terminus. ( C ) SUMOylation of FLAG-tagged NSP14 (SARS-CoV-2) WT or indicated mutants in transiently transfected HEK293T cells that were co-transfected for 40 h with either empty vector or V5-tagged TRIM32 together with HA-tagged UBC9 and Myc-tagged SUMO2, determined by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with the indicated antibodies. ( D ) Binding of FLAG-tagged NSP14 (SARS-CoV-2) WT or mutants in transiently transfected HEK293T cells that were co-transfected for 40 h with V5-tagged TRIM32, determined by PD: FLAG and IB with anti-V5. WCLs were probed by IB with the indicated antibodies. ( E ) Representation of the cryo-EM structure of the SARS-CoV-2 NSP10-NSP14-RNA complex (PDB: 7N0B) with NSP14 (cyan), RNA (orange), and NSP10 (green). K9 and K200 are depicted in red using spheres. Bound zinc ions are depicted as grey spheres. Data are representative of at least two independent experiments (C and D) or are from one unbiased mass spectrometry screen (A and B). See also Figure S3.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Mass Spectrometry, Transfection, Mutagenesis, Affinity Purification, SDS Page, Staining, Modification, Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: ExoN SUMOylation by TRIM32 inhibits RNA and NSP10 binding, suppressing ExoN activity ( A ) Schematic diagram of the in vitro exonuclease and RNA-binding assays to assess the effect of TRIM32-mediated SUMOylation on NSP14 ExoN activity and RNA-substrate binding. FLAG-tagged NSP14 or empty vector (control) was expressed in transiently transfected HEK293T cells together with either empty vector or TRIM32-V5 (WT, ΔRING or mutSIM) and Myc-SUMO2 and UBC9-HA, followed by affinity purification of NSP14-FLAG by PD: FLAG and subsequent elution using 3X-FLAG peptide. One portion of purified NSP14-FLAG was incubated with recombinant His-tagged NSP10 (rNSP10) and RNA substrate (CoV-RNA1-A) for the indicated times to analyze ExoN activity (lower center). Furthermore, eluted NSP14-FLAG was used for in vitro RNA binding assay with a biotinylated RNA substrate (CoV-RNA1-A) and subjected to PD: Strep and IB with anti-FLAG (lower right). The third portion of the eluted NSP14-FLAG fraction was analyzed by IB with anti-Myc to assess NSP14 SUMOylation (lower left). Parts of this figure were created using Biorender.com. ( B ) In vitro ExoN activity of purified SARS-CoV-2 NSP14 in the absence or presence of TRIM32 (WT or mutants) and the indicated SUMOylating machinery components, determined as described in (A). ( C ) Structural modeling of SARS-CoV-2 NSP14 with K9 and K200 SUMOylated. NSP10 (light green) and RNA (orange) from the NSP10-NSP14-RNA cryo-EM structure (PDB:7N0B) are depicted using surface representation showing steric clashes with SUMO molecules (K9-SUMO and K200-SUMO in NSP14 (cyan)). ( D ) RNA binding of SARS-CoV-2 NSP14-FLAG purified from mammalian cells co-expressing either empty vector or TRIM32 (WT or mutants) and the indicated SUMOylating machinery components and incubated with biotinylated CoV-RNA1-A (or control RNA), determined by PD: Strep and IB with anti-FLAG. ( E ) SARS-CoV-2 NSP14 binding to NSP10 in HEK293T cells that were co-transfected for 40 h with NSP14-FLAG, NSP10-Strep, UBC9-HA, Myc-SUMO2, and either empty vector or V5-tagged TRIM32 WT or mutants (ΔRING or mutSIM), determined by PD: FLAG and IB with anti-Strep. NSP14 SUMOylation was assessed in parallel by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with anti-FLAG, anti-Strep, anti-V5, anti-Myc, anti-HA, and anti-Actin (loading control). ( F ) SARS-CoV-2 replication in stable Caco-2 cells that inducibly (by DOX treatment (2 µg/mL) for 24 h) expressed FLAG-tagged TRIM32 and were transfected for 40 h with si.C, si.UBC9, or si.SUMO2, followed by infection with SARS-CoV-2 (strain WA1; MOI 0.02). At 48 h post-infection, SARS-CoV-2 g. Nsp3 and sg. N&Orf9b transcripts were determined by RT-qPCR. ( G ) Representative knockdown efficiency of endogenous UBC9 and SUMO2 for the experiment shown in (F), determined by RT-qPCR. Data (B, D, E, F, G) are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates (F and G). **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). ns, not significant. See also Figure S4.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Binding Assay, Activity Assay, In Vitro, RNA Binding Assay, Plasmid Preparation, Control, Transfection, Affinity Purification, Purification, Incubation, Recombinant, Cryo-EM Sample Prep, Expressing, Infection, Quantitative RT-PCR, Knockdown

Journal: bioRxiv

Article Title: Inhibition of coronaviral exonuclease activity by TRIM-mediated SUMOylation

doi: 10.1101/2025.07.28.667286

Figure Lengend Snippet: NSP14 SUMOylation by TRIM32 is broadly conserved for coronaviruses ( A ) SUMOylation of NSP14 from the indicated coronaviruses in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral NSP14s together with UBC9-HA and Myc-SUMO2 and either empty vector or V5-tagged TRIM32, assessed by PD: FLAG and IB with anti-Myc. WCLs were probed by IB with the indicated antibodies. ( B ) TRIM32 binding of the indicated coronaviral NSP14 proteins in HEK293T cells that were transiently transfected with plasmids expressing FLAG-tagged coronaviral NSP14s together with either empty vector or V5-tagged TRIM32, determined by PD: FLAG and IB with anti-V5. FLAG-tagged NSP10 (SARS-CoV-2) served as negative control. ( C ) MHV replication in stable Caco-2 cells that inducibly (by DOX treatment (2 µg/mL) for 24 h) expressed FLAG-tagged TRIM32 and were transfected for 40 h with si.C, si.UBC9 or si.SUMO2, followed by infection with MHV (strain A59; MOI 0.02) for 48 h, determined by RT-qPCR of g.N transcripts. ( D ) Representative knockdown efficiency of endogenous UBC9 and SUMO2 for the experiment in (C), determined by RT-qPCR. Data (A—D) are representative of at least two independent experiments (mean ± s.d. of n = 3 biological replicates (C and D). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student’s t test). See also Figure S4.

Article Snippet: Caco-2 TRIM32 -KO cells were generated by CRISPR-Cas9-mediated genome editing using a TRIM32 CRISPR/Cas9 KO plasmid (sc-402731; Santa Cruz).

Techniques: Transfection, Expressing, Plasmid Preparation, Binding Assay, Negative Control, Infection, Quantitative RT-PCR, Knockdown

Journal: Cell & Bioscience

Article Title: mTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A

doi: 10.1186/s13578-022-00919-y

Figure Lengend Snippet: c-Myc promotes TRIM32 mediated proteasomal degradation of SIRT4. A Western blot analysis of SIRT4 and c-Myc expression in the whole cellular lysates treated with c-Myc shRNA or MG132 for 48 h. B Western blot analysis of TRIM32 and c-Myc expression in whole cellular lysates treated with c-Myc shRNA. C SIRT4 interacts with TRIM32 in vivo. SNU449/HEK293 cell lysates were immunoprecipitated (IP) with control IgG, anti-SIRT4 or anti-TRIM32 antibodies, and then the precipitated proteins were detected by anti-TRIM32 or anti-SIRT4 antibodies, respectively. D SIRT4 degradation curves under various conditions. TRIM32 was knockdown with shRNA or forced overexpressed in SNU449 cells with or without being treated by proteasome inhibitors MG132 or lactacystin. After being treated with protein synthesis inhibitor cycloheximide (CHX; 50 μg/ml), the above cells were collected at the indicated hours. SIRT4 protein levels were detected by immunoblotting in the SNU449 total cell lysates. E SNU449 cells expressing FLAG-SIRT4 and co-expressing with or without hemagglutinin-tagged ubiquitin (HA-Ub) were co-transfected c-Myc and TRIM32 shRNA (left) or co-transfected c-Myc shRNA and TRIM32 (right) for 48 h. After being treated with 5 µM MG132 for 8 h, cells were collected and IP was performed by using FLAG antibody. Polyubiquitination of FLAG-SIRT4 were detected by anti HA-Ub antibody. F The indicated lysine sites of SIRT4 were mutated into arginine. G Lysates from SNU449 cells expressing the above SITR4 site mutants and co-expressing with or without hemagglutinin-tagged ubiquitin (HA-Ub) were pulled down with anti-FLAG, and then immunoblotted with anti HA-Ub antibody. Group differences were analyzed by two-tailed Student’s t test ( D ) (*p < 0.05, **p < 0.01)

Article Snippet: c-Myc shRNA (sc-29226-SH), TRIM32 siRNA (sc-61714), TRIM32 shRNA (sc-61714-SH), SIRT4 shRNA (sc-63024-SH), MAT2A shRNA (sc-106203-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, shRNA, In Vivo, Immunoprecipitation, Control, Knockdown, Ubiquitin Proteomics, Transfection, Two Tailed Test

Journal: Cell & Bioscience

Article Title: mTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A

doi: 10.1186/s13578-022-00919-y

Figure Lengend Snippet: Myc promoted SIRT4 degradation to reduce ADP ribosylation of MAT2A in the HCC. A Protein levels of c-Myc, MAT2A and MARylation level of MAT2A were determined by western blot from 85 pairs of tumour tissues. In the inserted boxplots, the circles indicate the median; the square indicate the 5th–95th percentiles. The statistical analysis was performed by the two tailed Student’s t-test (**p < 0.01). B Correlation between Myc and SIRT4 in HCC patients. 85 tissue pairs were immunohistochemically stained with indicated antibodies. The pair-wise Pearson correlation coefficient and the corresponding p-value between two genes were calculated. C The patients with high SIRT4 expression (n = 43) have longer survival compared to low SIRT4 expression (n = 42). Significance was determined using Kaplan–Meier analysis. D Schematic showing that high methionine content is sensed by mTORC1 that triggers c-Myc expression, which promotes TRIM32-mediated SIRT4 degradation. SIRT4 regulates MAT2A activity through MARylation, thereby inhibits MAT2A mediated tumour growth. SIRT4 loss resulted in the low MARylation level of MAT2A, which promotes methionine metabolism to generate SAM. In a feedback, high SAM level triggers transcription of metabolic enzymes to maintain tumour progression. Upward pointing arrows indicate an increase, and downwards pointing arrows indicate a decrease

Article Snippet: c-Myc shRNA (sc-29226-SH), TRIM32 siRNA (sc-61714), TRIM32 shRNA (sc-61714-SH), SIRT4 shRNA (sc-63024-SH), MAT2A shRNA (sc-106203-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Two Tailed Test, Staining, Expressing, Activity Assay

Journal: Cell & Bioscience

Article Title: mTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A

doi: 10.1186/s13578-022-00919-y

Figure Lengend Snippet: c-Myc promotes TRIM32 mediated proteasomal degradation of SIRT4. A Western blot analysis of SIRT4 and c-Myc expression in the whole cellular lysates treated with c-Myc shRNA or MG132 for 48 h. B Western blot analysis of TRIM32 and c-Myc expression in whole cellular lysates treated with c-Myc shRNA. C SIRT4 interacts with TRIM32 in vivo. SNU449/HEK293 cell lysates were immunoprecipitated (IP) with control IgG, anti-SIRT4 or anti-TRIM32 antibodies, and then the precipitated proteins were detected by anti-TRIM32 or anti-SIRT4 antibodies, respectively. D SIRT4 degradation curves under various conditions. TRIM32 was knockdown with shRNA or forced overexpressed in SNU449 cells with or without being treated by proteasome inhibitors MG132 or lactacystin. After being treated with protein synthesis inhibitor cycloheximide (CHX; 50 μg/ml), the above cells were collected at the indicated hours. SIRT4 protein levels were detected by immunoblotting in the SNU449 total cell lysates. E SNU449 cells expressing FLAG-SIRT4 and co-expressing with or without hemagglutinin-tagged ubiquitin (HA-Ub) were co-transfected c-Myc and TRIM32 shRNA (left) or co-transfected c-Myc shRNA and TRIM32 (right) for 48 h. After being treated with 5 µM MG132 for 8 h, cells were collected and IP was performed by using FLAG antibody. Polyubiquitination of FLAG-SIRT4 were detected by anti HA-Ub antibody. F The indicated lysine sites of SIRT4 were mutated into arginine. G Lysates from SNU449 cells expressing the above SITR4 site mutants and co-expressing with or without hemagglutinin-tagged ubiquitin (HA-Ub) were pulled down with anti-FLAG, and then immunoblotted with anti HA-Ub antibody. Group differences were analyzed by two-tailed Student’s t test ( D ) (*p < 0.05, **p < 0.01)

Article Snippet: c-Myc shRNA (sc-29226-SH), TRIM32 siRNA (sc-61714), TRIM32 shRNA (sc-61714-SH), SIRT4 shRNA (sc-63024-SH), MAT2A shRNA (sc-106203-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, shRNA, In Vivo, Immunoprecipitation, Control, Knockdown, Ubiquitin Proteomics, Transfection, Two Tailed Test

Journal: Cell & Bioscience

Article Title: mTORC1-c-Myc pathway rewires methionine metabolism for HCC progression through suppressing SIRT4 mediated ADP ribosylation of MAT2A

doi: 10.1186/s13578-022-00919-y

Figure Lengend Snippet: Myc promoted SIRT4 degradation to reduce ADP ribosylation of MAT2A in the HCC. A Protein levels of c-Myc, MAT2A and MARylation level of MAT2A were determined by western blot from 85 pairs of tumour tissues. In the inserted boxplots, the circles indicate the median; the square indicate the 5th–95th percentiles. The statistical analysis was performed by the two tailed Student’s t-test (**p < 0.01). B Correlation between Myc and SIRT4 in HCC patients. 85 tissue pairs were immunohistochemically stained with indicated antibodies. The pair-wise Pearson correlation coefficient and the corresponding p-value between two genes were calculated. C The patients with high SIRT4 expression (n = 43) have longer survival compared to low SIRT4 expression (n = 42). Significance was determined using Kaplan–Meier analysis. D Schematic showing that high methionine content is sensed by mTORC1 that triggers c-Myc expression, which promotes TRIM32-mediated SIRT4 degradation. SIRT4 regulates MAT2A activity through MARylation, thereby inhibits MAT2A mediated tumour growth. SIRT4 loss resulted in the low MARylation level of MAT2A, which promotes methionine metabolism to generate SAM. In a feedback, high SAM level triggers transcription of metabolic enzymes to maintain tumour progression. Upward pointing arrows indicate an increase, and downwards pointing arrows indicate a decrease

Article Snippet: c-Myc shRNA (sc-29226-SH), TRIM32 siRNA (sc-61714), TRIM32 shRNA (sc-61714-SH), SIRT4 shRNA (sc-63024-SH), MAT2A shRNA (sc-106203-SH) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Two Tailed Test, Staining, Expressing, Activity Assay

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 is selectively expressed in inner EGL and displays an uneven cytoplasmic distribution during GNP differentiation. a RT-qPCR analysis of Trim32 in P0, P7, P14, and adult mouse cerebella. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, and MycN in P0, P7, P14, and adult mouse cerebellums. c Immunofluorescence staining of Trim32 (red) in the EGL of P7 Math1-GFP transgenic mouse cerebellum. Nuclei were counterstained with DAPI (blue). EGL external granule layer, ML molecular layer, PCL Purkinje cells layer, and IGL internal granule layer. The scale bars represent 100 µm in the first panel and 25 µm in the second panel. d Confocal analysis (left) and quantification of immunofluorescence intensities (right) of distribution of Trim32 (green) in the dividing GNPs in different phases of the cell cycle. The dashed line highlights the cells that are in the indicated phase of cell cycle. The cell-cycle phases were identified by PH3 staining (red) for DNA. Nuclei were counterstained with DAPI (blue). The scale bars represent 10 µm. Data are expressed as means ± SD (n = 3). ns indicates P > 0.05 and triple asterisks indicate P < 0.001

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Transgenic Assay

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout enhances GNP proliferation in the postnatal developing cerebellum. a Expression of the Math1-GFP protein (GFP, green) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in a representing Math1 positive cells normalized to the length of the EGL edge. The scale bar represents 25 µm. Immunofluorescence staining of NeuN (red, b) and Ki67 (red, c) in P7 mouse cerebellar sections from the Math1-GFP/Trim32wt mice and Math1-GFP/Trim32KO mice. Nuclei were counterstained with DAPI (blue). Graph in b and c respectively representing NeuN or Ki67-positive cells normalized to the length of the EGL edge. The scale bar in b represents 50 µm. The scale bar in c represents 25 µm. oEGL outer external granule layer, iEGL inner external granule layer, ML molecular layer, and IGL internal granule layer. d P7 and P18 cerebellar midsagittal sections were stained for DAPI to show the overall morphology of cerebellum in Trim32wt mice and Trim32ko mice. The scale bar represents 500 µm

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: Knock-Out, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 antagonizes the SHH signaling activity. a RT-qPCR analysis of Trim32, the granule neuronal progenitor marker Maht1, and SHH target genes in P7 cerebellar granule neuronal progenitors (cGNPs) from Trim32wt mice and Trim32KO mice. Data are expressed as means ± SD (n = 3). Double asterisks indicate P < 0.01 and triple asterisks indicate P < 0.001. b Immunoblotting analysis of Trim32, Gli1, Ccnd1, Ccnd2, and MycN in P7 mouse cerebellum from Trim32wt mice and Trim32KO mice. c, d RT-PCR mRNA expression of the SHH target genes Gli1 and MycN in HEK293T cells overexpressing Trim32-GFP or GFP control vector. The cells were cultured with or without SHH for 24 and 48 h. Data are expressed as means ± SD (n = 3). Double asterisks indicate P < 0.01 and triple asterisks indicate P < 0.001. Gli-RE-luciferase activity in human medulloblastoma cell line D283 (e) and HEK293T cells (f), following transfected the indicated vectors. The luciferase activity was evaluated relative to Renilla activity. The means ± SD from three experiments are shown

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: Activity Assay, Quantitative RT-PCR, Marker, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, Plasmid Preparation, Cell Culture, Luciferase, Transfection

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 interacts with Gli1 though the NHL domain. a In vivo assay for binding between Trim32 and Gli1. Expression vectors encoding TRIM32-GFP and Flag-tagged Gli1 were transfected into HEK293T cells. Whole cell lysates were immunoprecipitated with anti-Trim32 or anti-FLAG antibody and immunoblotted with anti-Trim32 and anti-Gli1 antibodies. b Co-immunoprecipitation of exogenously expressed Trim32-HA with the endogenous Gli1 in HEK293T cells. c Co-immunoprecipitation of exogenously expressed Trim32-GFP with the Gli1 truncated mutants. d Schematic drawing of full-length Trim32 (Trim32-wt) and deletion mutants. Trim32 contains a RING finger (R), two B-boxes (B), a coiled-coil region (Coil), and an NHL domain (NHL). e Co-immunoprecipitation of exogenously expressed Flag-tagged Gli1 with the Trim32 truncated mutants shown as in d

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: In Vivo, Binding Assay, Expressing, Transfection, Immunoprecipitation

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 promotes Gli1 ubiquitination and degradation. a HEK293T cells transfected with Gli1 and increasing doses of Trim32 were collected for immunoblotting with the indicated antibodies. b Quantification of Gli1 and Trim32 protein levels (normalized to actin) from a. The means ± SEM from three experiments are shown. c Cells stably expressing scramble RNA or shRNA targeting Trim32 (#1, #2) were harvested for immunoblotting with the indicated antibodies. d HEK293T cells were transfected with plasmids encoding Flag-tagged Gli1 and control GFP vector or Trim32 or Trim32-ΔR, treated with 20 µg/ml cycloheximide (CHX) and harvested at the indicated times points. The levels of Gli1 and Trim32 in the lysates were investigated by immunoblotting. e Quantification of Gli1 remaining protein levels (normalized to actin) from d. The means ± SEM from three experiments are shown. f Cells were transfected with scramble RNA or shRNA targeting Trim32 (#1), treated with 20 µg/ml cycloheximide (CHX), collected at the indicated time points, and then immunoblotted with the indicated antibodies. g Quantification of Gli1 protein levels (normalized to actin) from f. The means ± SEM from three experiments are shown. h Cells transfected with Gli1 or Trim32 were treated with or without 10 µM MG132 for 8 h, collected, and then immunoblotted with the indicated antibodies. Graph representing quantification of Gli1 protein levels (normalized to actin). i Anti-ubiquitin immunoblotting of immunoprecipitated exogenous Gli1 in HEK293T cells transfected with the indicated plasmids. j The effect of Trim32-wt or Trim32 deletion mutant (Trim32-ΔR) to the activation of Gli-RE-luciferase induced by Gli1 in HEK293T cells. The means ± SD from three experiments are shown. Double asterisks indicate P < 0.01

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: Ubiquitin Proteomics, Transfection, Western Blot, Stable Transfection, Expressing, shRNA, Control, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Activation Assay, Luciferase

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: Trim32 knockout increases the incidence of MB in Ptch1+/− mice. Quantitative PCR mRNA (a; mean ± SD; n = 9) and protein levels (b; n = 3) of Trim32 in mouse medulloblastomas (MB) from Ptch1+/− mice and normal cerebella. c RNA expression level of Trim32 negatively correlated with Gli1 (n = 51) in human SHH MB samples. Data are analyzed from Oncomine database. d Kaplan–Meier analysis of MB incidence in 63 Ptch1+/−/Trim32wt mice (gray line) versus 17 Ptch1+/−/Trim32KO mice (black line). Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice, obtained by interbreeding for at least three generations the progeny of Ptch1 heterozygous and Trim32 knock-out mice, were then monitored for the onset of medulloblastoma; (triple asterisks indicate P < 0.001, Logrank test). e Expression of the Math1-GFP protein (GFP, green) in the Math1-GFP mouse medulloblastomas and adjacent cerebellar cortices sections from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Nuclei were counterstained with DAPI (blue). The scale bar represents 200 µm. MB medulloblastoma, IGL internal granule layer. RT-qPCR analysis of SHH target genes (f), Trim32 (g), the granule neuronal progenitor marker Maht1 (h), and the differentiated granule cell markers including Tuj1 and NeuN (i) in adult mouse cerebellums and medulloblastomas from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice. Data are expressed as means ± SD (n = 3). An asterisk indicates P < 0.05, double asterisks indicate P < 0.01, and triple asterisks indicate P < 0.001. j Immunoblotting analysis of Trim32, Gli1, Ccnd2, MycN, and NeuN in MB from Ptch1+/−/Trim32wt mice and Ptch1+/−/Trim32KO mice

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Quantitative RT-PCR, Marker, Western Blot

Journal: Cell Death and Differentiation

Article Title: Trim32 suppresses cerebellar development and tumorigenesis by degrading Gli1/sonic hedgehog signaling

doi: 10.1038/s41418-019-0415-5

Figure Lengend Snippet: mRNA expression analysis

Article Snippet: The shRNA of Trim32 was ordered from OriGene (#TL308646), and the sequences of shRNA were as followed: #1: 5′-GTCCAATAGTCAAGTGGTAGAGGAGCAGA-3′; #2: 5′-AGGCAGATGTAGCACTACTGGAGGAGACA-3′.

Techniques: Expressing