Journal: Brain

Article Title: Dysregulation of protein SUMOylation networks in Huntington’s disease R6/2 mouse striatum

doi: 10.1093/brain/awae319

Figure Lengend Snippet: mGLUR7 SUMOylation and endocytosis . ( A ) SUMO-limiting experiment in HeLa cells transfected with His-SUMO1, Myc- mGLUR7 and PIAS1. Top : His-pulldown of His-SUMO1 protein. Bottom : Trichloroacetic acid protein precipitation (TCA) whole-cell lysate. Full SUMO transfections (e.g. ‘+’) included 2 µg of SUMO1 per replicate, whereas one-quarter SUMO1 (e.g. ‘1/4’) were transfected with 0.5 µg of SUMO1 plasmid. The area outlined in orange is an example of the area used to quantify each lane. The full blot is provided in . ( B ) Quantification of the Myc-mGLUR7 signal is shown in the His-isolated ( left ) and whole-cell lysate ( right ) conditions. His-isolated samples showed a significant difference in levels of SUMO-mGLUR7 isolated. F (4,9) = 34.42, P < 0.0001 with Šídák’s multiple comparisons test: mGLUR7 versus mGLUR7 + SUMO1, P adj = 0.0173; mGLUR7 + SUMO1 versus mGLUR7 + 1/4 SUMO1, P adj = 0.0165; mGLUR7 + 1/4 SUMO1 versus mGLUR7 + 1/4 SUMO1 + PIAS1, P adj = 0.0001; mGLUR7 + SUMO1 versus mGLUR7 + 1/4 SUMO1 + PIAS1, P adj = 0.0190; analysed by one-way ANOVA. TCA samples show no change in mGLUR7 levels. One-way ANOVA with Šídák’s multiple comparisons test: F (4,9) = 0.6085, P = 0.6668. n = 2 for His-SUMO1-only control transfections, n = 3 transfections for all other conditions. ( C ) Schematic of mGLUR7 internalization experiment. Created in BioRender ( BioRender.com/i50e678 ). ( D ) The endocytosis of mGLUR7 was assessed by antibody uptake internalization assay. Non-transgenic (NT) and Huntington’s disease (HD) primary striatal neurons (PSNs) from R6/2 pups were treated with Pias1 or control siRNA, transfected with Myc-tagged mGLUR7, labelled with anti-c-Myc antibody, washed and returned to conditioned media at 37°C for 15 min. Representative images are of transfected mGLUR7 receptor internalization. Internalized receptors, green; surface receptors, magenta (false-coloured from red for clarity); nuclei, blue. The internalization ratio was the fluorescence intensity of the internalized receptor (green) over the total amount of receptor (sum of green and red intensity shown in magenta). Individual channels are shown in . ( E ) Receptor internalization assay for mGLUR7 in R6/2 primary striatal neurons (PSNs) demonstrated a significant decrease in mGLUR7 internalization in HD neurons compared to NT cells. Treatment by Pias1 siRNA-mediated knockdown significantly reversed levels of mGLUR7 internalization. mGLUR7 internalization: treatment, F (1,29) = 18.81, P = 0.0002; genotype, F (1,29) = 0.001509, P = 0.9693; analysed by two-way ANOVA. * P < 0.05, *** P < 0.001, **** P < 0.0001, n = branch areas, three per neuron. Graphs represent mean ± standard error of the mean.

Article Snippet: Western blot analysis was used to determine the quantitative levels of SUMO1 (ENZO, Cat. No. BML-PW8330), SUMO2 (ENZO, Cat. No. BML-PW0510A), PIAS1 (Cell Signaling, Cat. No. D33A7 XP), mGLUR7 (EMD Millipore, Cat. No. 07-239) and Myc-tag (Millipore Sigma, Cat. No. 05-419) using specific antibodies according to our previously published protocol and in the .

Techniques: Transfection, Plasmid Preparation, Isolation, Control, Transgenic Assay, Fluorescence, Knockdown

Journal: Brain

Article Title: Dysregulation of protein SUMOylation networks in Huntington’s disease R6/2 mouse striatum

doi: 10.1093/brain/awae319

Figure Lengend Snippet: PIAS1 knockdown rescues alterations to neuronal activity in Huntington’s disease primary cortical neurons . ( A ) Experimental schematic of microelectrode array (MEA) workflow. Created in BioRender ( BioRender.com/s52a685 ). ( B ) Significant differences identified in various MEA metrics at three time points [14, 16 and 19 days in vitro (DIV14, DIV16 and DIV19)] between conditions: Genotype effect [significant difference between non-transgenic (NT) control knockdown and Huntington’s disease (HD) control knockdown cells], PIAS1 knockdown effect in HD cells (significant difference between control knockdown and PIAS1 knockdown in HD cells) and PIAS1 knockdown effect in NT cells (significant difference between control knockdown and PIAS1 knockdown in NT cells). Blue or yellow indicates significantly decreased or increased, respectively, values in the HD cells (compared to NT cells) and PIAS1 knockdown cells [compared to control siRNA (in either HD or NT cells)], respectively. ‘X’ indicates no significant difference detected. ( C ) Weighted mean firing rate (WMFR) was significantly higher in control knockdown-treated HD cells compared to control knockdown-treated NT cells at DIV14 and DIV16. This pattern reversed at DIV19. The increases seen in the WMFR in HD at DIV14 and DIV16 were significantly reduced upon knockdown with Pias1 siRNA. Primary cortical neurons (PCNs) DIV14 WMFR: treatment, F (1,20) = 0.8446, P = 0.3690, genotype, F (1,20) = 12.11, P = 0.0024; PCN DIV 16 WMFR: treatment, F (1,20) = 13.63, P = 0.0014, genotype, F (1,20) = 14.75, P = 0.0010; PCN DIV 19 WMFR: treatment, F (1,20) = 1.243, P = 0.2781, genotype, F (1,20) = 2.793, P = 0.1102. * P < 0.05, *** P < 0.001. Graphs represent means ± standard error of the mean analysed by two-way ANOVA. A master analysis sheet of all significant differences in the remaining time points of PCNs can be found in the , File 016. n = 6 wells/condition.

Article Snippet: Western blot analysis was used to determine the quantitative levels of SUMO1 (ENZO, Cat. No. BML-PW8330), SUMO2 (ENZO, Cat. No. BML-PW0510A), PIAS1 (Cell Signaling, Cat. No. D33A7 XP), mGLUR7 (EMD Millipore, Cat. No. 07-239) and Myc-tag (Millipore Sigma, Cat. No. 05-419) using specific antibodies according to our previously published protocol and in the .

Techniques: Knockdown, Activity Assay, Microelectrode Array, In Vitro, Transgenic Assay, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUMOylation of SETD8 Promotes Tumor Growth by Methylating and Stabilizing MYC in Bladder Cancer.

doi: 10.1002/advs.202501734

Figure Lengend Snippet: Figure 5. SUMOylation enhances SETD8 protein stability. a) Genes ranked at the top that regulate SETD8 stability are shown via MAGeCK analysis. The X-axis represents the ranking, while the Y-axis indicates the robust rank aggregation (RRA) score for each gene. b) T24 cells treated with TAK-981 (1 μM, 6 h) were subjected to co-IP assay to detect endogenous SUMOylation level of SETD8 via western blotting. c) Time-course analysis of SETD8 protein in T24 cells incubated with TAK-981 (1 μM, 6 h), followed by the addition of CHX (20 μg mL−1) at the indicated time point. d) Immunoblot analysis of exogenous SUMOylation level of SETD8 mediated by PIAS1 in HEK-293T cells upon the treatment of TAK-981 (1 μM, 6 h). e) Immunoblot analysis of SUMOylation level of ectopic SFB-SETD8 WT and K219R mutant in HEK-293T cells. f) Immunoblot analysis of ubiquitination level of SFB-SETD8 WT and K219R mutant in HEK-293T cells treated with MG132 (10 μM, 6 h). g) Time-course analysis in HEK-293T cells expressing SFB-SETD8 WT or K219R mutant following treatment with CHX (20 μg mL−1). h) Immunoblot analysis of SUMOylation level of SFB-SETD8 when co-transfected with 3xMYC-SENP6 in HEK-293T cells. Statistical significance was determined from three independent experiments. Data are presented as means ± SD, with p values calculated using Student’s t test.

Article Snippet: After blocking with 5% nonfat milk at room temperature for 1 h, the immunoblots were performed referring to the standard process using the indicated primary antibodies against Flag (CST, 14 793), HA (CST, 3724), V5 (CST, 13 202), MYC-tag (CST, 2278), SETD8 (CST, 2996), Mono-methyl lysine (Abnova, MAB12422), GAPDH (Fudebiotech, FD0063), GFP (CST, 2956), β-Tubulin (CST, 2146), SUMO2/3 (CST, 4971), PIAS1 (CST, 3550), and MYC (Abcam, ab32072).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Incubation, Mutagenesis, Ubiquitin Proteomics, Expressing, Transfection

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUMOylation of SETD8 Promotes Tumor Growth by Methylating and Stabilizing MYC in Bladder Cancer.

doi: 10.1002/advs.202501734

Figure Lengend Snippet: Figure 6. SUMOylated SETD8 enhances MYC-K412 methylation. a) Co-IP of V5-MYC and SFB-SETD8 WT or its K219R mutant in HEK-293T cells, in the presence of GFP-PIAS1 and HA-SUMO3. b) Co-IP of V5-MYC and SFB-SETD8 WT or its K219R mutant added with 1x, 2x, or 4x SUMO3. c) SIM domains in MYC predicted by JASSA (www.jassa.fr) and BIOCUCKOO (www.biocuckoo.org). d) Co-IP of HA-SETD8 and SFB-MYC mutants with synonymous mutations in predicted SIM motifs in HEK-293T cells. e) Immunoblot analysis of methylation level of SFB-MYC when co-expressed with SETD8 WT or K219R in HEK-293T cells. f) Immunoblot analysis of lysine methylation level of SFB-MYC with SIM mutation while co-transfected with SETD8 in HEK- 293T cells. g) Immunoblot analysis of ubiquitination level of SFB-MYC WT and SIM4 mutant in HEK-293T cells following MG132 (10 μM, 6 h) treatment. h) Time-course analysis was performed in HEK-293T cells expressing SFB-MYC WT or SIM4 mutant, with or without SETD8, following treatment with CHX (20 μg mL−1). Statistical significance was determined from three independent experiments. Data are presented as means ± SD, with p values calculated using Student’s t test.

Article Snippet: After blocking with 5% nonfat milk at room temperature for 1 h, the immunoblots were performed referring to the standard process using the indicated primary antibodies against Flag (CST, 14 793), HA (CST, 3724), V5 (CST, 13 202), MYC-tag (CST, 2278), SETD8 (CST, 2996), Mono-methyl lysine (Abnova, MAB12422), GAPDH (Fudebiotech, FD0063), GFP (CST, 2956), β-Tubulin (CST, 2146), SUMO2/3 (CST, 4971), PIAS1 (CST, 3550), and MYC (Abcam, ab32072).

Techniques: Methylation, Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Transfection, Ubiquitin Proteomics, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: SUMOylation of SETD8 Promotes Tumor Growth by Methylating and Stabilizing MYC in Bladder Cancer.

doi: 10.1002/advs.202501734

Figure Lengend Snippet: Figure 7. Model of the regulation of SETD/MYC axis in BC. SETD8 methylates MYC at K412, which prevents the CHIP-mediated degra- dation of MYC and promotes MYC accumulation and bladder tumor growth. SUMOylation of SETD8, modulated by PIAS1 and SENP6, sta- bilizes SETD8 and further strengthens its interaction with MYC through the SUMO-SIM interaction, leading to increased MYC methylation and enhanced tumor growth. Targeting the SETD8/MYC axis with SETD8 in- hibitor UNC0379 may provide a potential therapeutic strategy for BC pa- tients.

Article Snippet: After blocking with 5% nonfat milk at room temperature for 1 h, the immunoblots were performed referring to the standard process using the indicated primary antibodies against Flag (CST, 14 793), HA (CST, 3724), V5 (CST, 13 202), MYC-tag (CST, 2278), SETD8 (CST, 2996), Mono-methyl lysine (Abnova, MAB12422), GAPDH (Fudebiotech, FD0063), GFP (CST, 2956), β-Tubulin (CST, 2146), SUMO2/3 (CST, 4971), PIAS1 (CST, 3550), and MYC (Abcam, ab32072).

Techniques: Methylation