Journal: PLoS ONE

Article Title: Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

doi: 10.1371/journal.pone.0268835

Figure Lengend Snippet: Effects of TAK243 (ubiquitination inhibitor) and ML792 (SUMOylation inhibitor) on the number and size of PML–NBs in CHOGFPPML (A) and HEKGFPPML cells (B). The cells were cultured in the presence of 0.1% DMSO (control), 10 μM TAK243 for 4 h, or 20 μM ML792 for 4 and 24 h. The number of PML–NBs in both cell types decreased and their sizes increased significantly by treatment with ML792. TAK243 was less effective than ML792 in decreasing the number of PML–NBs, and the cells looked shrunken and slightly damaged after 6 h of culture with 10 μM TAK243. Data are presented as the mean ± SEM. *, Significantly different from control group. #, Significantly different from all other groups. &, Significantly different from control and TAK243 groups. Scale bar = 10 μm.

Article Snippet: TAK243 (Ub-activating E1 enzyme inhibitor, Selleckchem, Houston, TX) and ML792 (SUMO E1 inhibitor, MedKoo Bioscience, Morrisville, NC) were dissolved in DMSO and used at a final concentration of 0.1% DMSO.

Techniques: Ubiquitin Proteomics, Cell Culture, Control

Journal: PLoS ONE

Article Title: Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

doi: 10.1371/journal.pone.0268835

Figure Lengend Snippet: Effects of TAK243 and ML792 on As 3+ –induced biochemical changes of PML in CHOGFPPML (A) and HEKGFPPML cells (B). The cells were pre–treated with 0.1% DMSO, 10 μM TAK243, or 20 μM ML792 for 3 h, then further treated with 3 μM As 3+ or left untreated for 2 h. The RIPA–soluble and–insoluble fractions were prepared as described previously, and the proteins were resolved by SDS–PAGE followed by western blotting. For CHOGFPPML cells, samples following each treatment were obtained from 2 separate wells. The membranes were probed sequentially with the indicated antibodies, then reprobed with a mixture of HRP–tagged anti–tubulin and HRP–tagged anti–histone H3 antibodies. Open arrow, mono–ubiquitinated histone; Closed arrow, GFPPML; Open arrowhead, SUMO2/3 monomers; Closed arrowhead, SUMO1 monomers.

Article Snippet: TAK243 (Ub-activating E1 enzyme inhibitor, Selleckchem, Houston, TX) and ML792 (SUMO E1 inhibitor, MedKoo Bioscience, Morrisville, NC) were dissolved in DMSO and used at a final concentration of 0.1% DMSO.

Techniques: SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Effects of arsenic on the topology and solubility of promyelocytic leukemia (PML)-nuclear bodies

doi: 10.1371/journal.pone.0268835

Figure Lengend Snippet: The cells were treated with 0.1% DMSO or 20 μM ML792 for 3 h, then further treated with 3 μM As 3+ or left untreated for 2 h. Note that SUMO2/3 in peri–nuclear PML aggregates (arrow) was decreased in the DMSO–treated cells, whereas a substantial amount of SUMO2/3 retained with GFPPML in the ML792–treated cells. Scale bar = 10 μm.

Article Snippet: TAK243 (Ub-activating E1 enzyme inhibitor, Selleckchem, Houston, TX) and ML792 (SUMO E1 inhibitor, MedKoo Bioscience, Morrisville, NC) were dissolved in DMSO and used at a final concentration of 0.1% DMSO.

Techniques:

Journal: Cancers

Article Title: SUMOylation Is Associated with Aggressive Behavior in Chondrosarcoma of Bone

doi: 10.3390/cancers13153823

Figure Lengend Snippet: The SUMO E1 inhibitor ML792 blocks SUMO conjugation. ( A ) Cartoon of the function of the SUMO E1 inhibitor ML792. ( B ) Western blot analysis of SUMO2/3 and ubiquitin levels of the chondrosarcoma cell lines CH2879, JJ012, and NDCS1 treated with the indicated concentrations of the SUMO E1 inhibitor ML792 for 4 h compared to solvent control (DMSO 0.1%). Ponceau S staining was used as loading control. ( C ) Quantitative analysis of SUMO2/3 and ubiquitin conjugation of the corresponding western blots shown in B. Data represent mean with standard deviation ( n = 3).

Article Snippet: ML792 [ ] was obtained from Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Tokio, Japan), and was dissolved in DMSO for in vitro usage.

Techniques: Conjugation Assay, Western Blot, Ubiquitin Proteomics, Solvent, Control, Staining, Standard Deviation

Journal: Cancers

Article Title: SUMOylation Is Associated with Aggressive Behavior in Chondrosarcoma of Bone

doi: 10.3390/cancers13153823

Figure Lengend Snippet: Inhibition of SUMO E1 via ML792 results in decreased proliferative capacity and cell viability. ( A ) Two-dimensional (2D) colony-formation assay (crystal violet). CH2879, JJ012, and NDCS1 cells were treated with ML792 cells using single treatment or repetitive treatment on day 1, 4, 7, and 10 after seeding. Colony-formation was quantified via measuring crystal violet staining. Data represent mean with standard deviation ( n = 3). ( B ) Viability assay using PrestoBlue. Chondrosarcoma cell lines were treated with ML792 and incubated for 3 days. Relative cell viability is represented as mean with standard deviation ( n = 3). ( C ) Expression levels of c-MYC, UBA2, UBC9 and conjugation of SUMO2/3 in lysates of untreated chondrosarcoma cell lines from C. Ƴ -tubulin was used as loading control. Single representative images are shown ( n = 3). Whole blots including intensity readings can be found in ( D ) Table displaying specifics of the cell lines from B including IC50 (nM) values. ( E ) Boxplots to compare IC50 values of dedifferentiated ( n = 3) and grade 2/3 ( n = 4) chondrosarcoma cell lines. Significance was calculated with a two-tailed t -test *: p < 0.05.

Article Snippet: ML792 [ ] was obtained from Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Tokio, Japan), and was dissolved in DMSO for in vitro usage.

Techniques: Inhibition, Colony Assay, Staining, Standard Deviation, Viability Assay, Incubation, Expressing, Conjugation Assay, Control, Two Tailed Test

Journal: Cancers

Article Title: SUMOylation Is Associated with Aggressive Behavior in Chondrosarcoma of Bone

doi: 10.3390/cancers13153823

Figure Lengend Snippet: SUMO E1 inhibition leads to G2/M arrest and chromosome bridge formation. ( A ) Cell cycle analysis by flow cytometry of chondrosarcoma cell lines treated with ML792 for 24 h. Western blot analysis confirmed the inhibition of SUMO conjugation by the SUMO E1 inhibitor ML792. ( B ) Data representation of A in bar-graphs indicating G1, S, and G2/M pool of the cell lines for 0 or 24 hours of treatment as mean with standard deviation ( n = 3). ( C ) Immunofluorescence images of a-synchronous CH2879, NDCS1, and JJ012 cells treated with ML792 for 24 h. Cells were stained with Hoechst to visualize DNA. Green arrows indicate the location of chromosome bridges. Insert: magnification of a chromosome bridge. Scale bars represent 10 µm. ( D ) Quantification of chromosome bridges in CH2879 cells upon treatment with the indicated concentration of the SUMO E1 inhibitor (ML792) for 24 and 48 h. For each replicate 15 images (approximately 200 cells total) per condition were analyzed. Data represent mean with standard deviation ( n = 3). ( E ) Quantification of micronuclei present in CH2879 cells upon treatment with the indicated concentration of the SUMO E1 inhibitor (ML792) for 24 and 48 h. For each replicate 15 images (approximately 200 cells total) per condition were analyzed. Data represents mean with standard deviation ( n = 3).

Article Snippet: ML792 [ ] was obtained from Millennium Pharmaceuticals, Inc. (Cambridge, MA, USA), a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Tokio, Japan), and was dissolved in DMSO for in vitro usage.

Techniques: Inhibition, Cell Cycle Assay, Flow Cytometry, Western Blot, Conjugation Assay, Standard Deviation, Immunofluorescence, Staining, Concentration Assay

Journal: The EMBO Journal

Article Title: HDAC6-dependent deacetylation of SAE2 enhances SUMO1 conjugation for mitotic integrity

doi: 10.1038/s44318-025-00532-y

Figure Lengend Snippet: ( A ) Representative western blot of SAE2 expression in stable, inducible siRNA-resistant SAE1:SAE2 variant U2OS cells. Cells were treated for 72 h with siRNA for either NTC or SAE1:SAE2 with the concurrent addition of 4 μg/ml Doxycycline to induce expression of the indicated integrated SAE1:2 constructs. Replicated >5 times in the laboratory. ( B ) Representative images depicting the localisation of SAE2 in interphase, prophase and metaphase U2OS cells. Cells depleted for SAE1:SAE2 and complemented with WT or indicated SAE1:SAE2-variants. Interphase cells received no synchronisation. Prophase and metaphase cells were synchronised in nocodazole for 16 h and either fixed immediately or after a 35 min release into mitosis, respectively. Five-micrometer scale bar is shown as a red line. Cells chosen from a representative field >20 similar cells. Performed once. ( C ) U2OS depleted for SAE1:SAE2 and complemented with WT or indicated SAE1:SAE2-variants, subjected to 43 °C for 40 min before replating and counting after colony growth. Significance calculated using one-way ANOVA. Error bars = SEM; N = 3 biological repeats. * = P ≤ 0.05, ns = not significant p > 0.05. Statistical values for NTC vs siSAE P = 0.0060, siSAE vs siSAE+SAE2-WT P = 0.0064, NTC vs siSAE+SAE2-WT P > 0.9999, siSAE+SAE2-WT vs siSAE+SAE2-C173G P = 0.0174, siSAE+SAE2-WT vs siSAE+SAE2-K164Q P = 0.9665, siSAE+SAE2-WT vs siSAE+SAE2-K164R P = 0.4874. ( D ) Automated analysis of γH2AX foci numbers, obtained through high-content microscopy, in U2OS cells treated with indicated siRNAs (siNTC- black, siSAE1:SAE2- red) with or without the complementation of inducible siRNA-resistant SAE2 variants (SAE2 WT- dark blue, SAE2 CG- orange, SAE2 K164Q- grey, SAE2 K164- light blue). siUBC9 (purple) and siPIAS1 (green) are used for comparison. Results displayed for data isolated from S phase (top), G1 (middle) and G2 (bottom) cell populations. Plotted data is derived from the mean number of foci per condition from 3 independent biological repeats, error bars = SEM. Statistical significance was calculated using two-way ANOVA using Dunnett’s multiple comparisons test. Timepoints where there is a significant difference from the non-target control siRNA condition are marked with * = P < 0.05, ** = P < 0.01, *** = P < 0.001. Purple and green * show that only siUBC9 and siPIAS1 conditions significantly deviate from siNTC at points in the time course. siNTC vs siSAE1:SAE2 (S phase 1 h P = 0.8997, 6 h P = 0.7437; G1 1 h P = 0.7623, 6 h P = 0.9997; G2 1 h P = 0.9993, 6 h P = 0.9988), siNTC vs SAE2-WT (S phase 1 h P = 0.9792, 6 h P > 0.9999; G1 1 h P = 0.9997, 6 h P = 0.7655; G2 1 h P = 0.9971, 6 h P = 0.7660), siNTC vs SAE2-CG (S phase 1 h P = 0.9907, 6 h P > 0.9999; G1 1 h P = 0.9965, 6 h P = 0.9989; G2 1 h P = 0.9957, 6 h P = 0.9494), siNTC vs SAE2-KQ (S phase 1 h P = 0.9686, 6 h P = 0.9993; G1 1 h P = 0.9978, 6 h P = 0.9932; G2 1 h P > 0.9999, 6 h P > 0.9999), siNTC vs SAE2-KR (S phase 1 h P = 0.8283, 6 h P > 0.9211; G1 1 h P = 0.9430, 6 h P = 0.9799; G2 1 h P = 0.9999, 6 h P = 0.9967), siNTC vs siUBC9 (S phase 1 h P = 0.0076, 6 h P = 0.0086; G1 1 h P = 0.0277, 6 h P = 0.9833; G2 1 h P = 0.0073, 6 h P = 0.5598), siNTC vs siPIAS1 (S phase 1 h P = 0.0016, 6 h P < 0.0001; G1 1 h P = 0.0002, 6 h P = 0.0020; G2 1 h P = 0.0002, 6 h P = 0.0051). ( E ) The measure of DNA repair from U2OS cells bearing integrated DNA repair reporters in cells treated with siNTC or siSAE1:siSAE2 and transfected with the enzyme, I-SCE-1. Illustration of the integrated DNA repair substrates for homologous recombination and non-homologous end-joining (Top). The graph (Bottom) displays the percentage of GFP-positive cells normalised to RFP-transfection efficiency. %-repair of siSAE1:SAE2 is given relative to siNTC. Data from 2 independent biological repeats. ( F ) Immunoprecipitation of endogenous mitotic SUMO conjugates in U2OS cells treated with ML792 and expressing Flag-SAE2 constructs resistant to the inhibitor. ML792 resistance is denoted by (r). The presence of Flag-SAE2r is represented by (WTr), Flag-SAE2r-K164Q by KQr, and Flag-SAE2r-K164R by KRr. The diagram, top, illustrates the timing of inhibitor and induction agent addition. To better detect free SUMO, Y299 (SUMO1) and 8A2 (SUMO2/3) antibodies were employed (Garvin et al, ). Performed once. ( G ) The percentage of micronuclei positive for γH2AX in asynchronous siRNA-resistant SAE2 variant-expressing U2OS cells. Error bars SEM. Data from 4 independent biological repeats. N > 50 micronuclei per condition per biological repeat. Significance was tested using one-way ANOVA no significant differences between conditions were identified. ( H ) Western blot analysis of U2OS treated with nocodazole ±5 µM ML792 for 16 h. Mitotic cells were harvested by mitotic shake-off and lysed in loading buffer, and western probed for NuMA and SAE2. Performed twice. ( I ) Representative images validating the specificity of the NuMA antibody. NuMA colocalises to -β tubulin metaphase cells adjacent to the DAPI stain. NuMA signal significantly diminished after 72-h 10 nM siNuMA treatment. Cells chosen from >50 similar cells, performed once. White bar indicates 5 micrometers.

Article Snippet: ML792 , Cambridge Bioscience , HY-108702.

Techniques: Western Blot, Expressing, Variant Assay, Construct, Microscopy, Comparison, Isolation, Derivative Assay, Control, Transfection, Homologous Recombination, Non-Homologous End Joining, Immunoprecipitation, Staining

Journal: The EMBO Journal

Article Title: HDAC6-dependent deacetylation of SAE2 enhances SUMO1 conjugation for mitotic integrity

doi: 10.1038/s44318-025-00532-y

Figure Lengend Snippet: ( A ) Immunoprecipitation of endogenous mitotic SUMO conjugates in U2OS cells treated with 1 μM ML792 and expressing FLAG-SAE2 constructs resistant to the inhibitor. ML792 resistance is denoted by (r). The presence of FLAG-SAE2r is represented by (WTr), FLAG-SAE2r-K164Q by K164Qr, and FLAG-SAE2r-K164R by K164Rr. Diagram, top, illustrates the timing of inhibitor and induction agent addition. Antibodies for SUMO proteins were EP298 (SUMO1) and 8A2 (SUMO2/3). Performed once. ( B ) Representative images of immunofluorescent analysis of mitotic spindle formation. Five micrometer scale bar is shown as a white line. ( C ) The percent of laterally presented metaphase and anaphase U2OS cells exhibiting multipolar spindles in cells complemented with SAE2 variants in RNAi-treated cells, as shown. Error bars SEM; significance calculated using one-way ANOVA, * P ≤ 0.05, ** P ≤ 0.01, ns = not significant >0.05. siSAE:1:SAE2 vs WT P = 0.0142, siSAE:1:SAE2 vs CG P = 0.9034, siSAE:1:SAE2 vs KQ P = 0.9964, siSAE:1:SAE2 vs KR = 0.0031. Data from three independent biological repeats. N > 50 cells per condition analysed from a minimum of four fields of view per biological repeat. ( D ) The percent of laterally presented metaphase and anaphase U2OS cells exhibiting multipolar spindles. Cells exposed to different durations of 1 μM ML792 prior to release from 0.332 µM nocodazole: long treatment totalling 20 h (added 4 h before nocodazole) is displayed to the left-hand side, short treatment totalling 1 h (added during the last hour of nocodazole), presented to the right-hand side. 1 μM ML792 was re-added to cells after release from nocodazole. Error bars SEM; significance calculated using one-way ANOVA, *** P ≤ 0.001, ns = not significant >0.05. ML792 20 h vs WTr P = 0.0002, ML792 20 h vs CGr P > 0.9999, ML792 20 h vs KQr P = 0.9982, ML792 20 h vs KRr P < 0.0001. ML792 1 h vs WTr P < 0.0001, ML792 1 h vs CGr P = 0.8286, ML792 1 h vs KQr P = 0.6389, ML792 1 h vs KRr P < 0.0001. Data from 3 independent biological repeats. N > 50 cells per condition from a minimum of four fields of view per biological repeat. ( E ) The percent of laterally presented metaphase and anaphase U2OS cells exhibiting multipolar spindles in cells treated with 1 μM ML792 and complemented with WTr, FLAG-SAE2r-K164Q, and FLAG-SAE2r-K164R with and without the addition of HDAC6 inhibitor. Error bars SEM; significance calculated using one-way ANOVA, ** P ≤ 0.01, ns = not significant >0.05. WTr + vehicle vs WTr + HDAC6i P = 0.0050, KQr + vehicle vs KQr + HDAC6i P = 0.9998, KRr + vehicle vs KRr + HDAC6i P = 0.9883. Data from four independent biological repeats. N > 50 cells per condition from a minimum of four fields of view per biological repeat. ( F ) Analysis of micronuclei in asynchronous siRNA-resistant SAE2 variant U2OS cells. The percentage of total cells with one or more micronuclei is plotted (Top). Error bars SEM; significance calculated using one-way ANOVA, * P ≤ 0.05, **≤0.01, ns = not significant >0.05. Data from three independent biological repeats. Cells from at least 4 fields of view were analysed. N > 400 total cells per condition per biological repeat. siSAE:1:SAE2 vs WT P = 0.0083, siSAE:1:SAE2 vs CG P = 0.9893, siSAE:1:SAE2 vs KQ P = 0.7940, siSAE:1:SAE2 vs KR P = 0.0176. The percentage of micronuclei positive for CENPA in asynchronous siRNA-resistant SAE2 variant cells. (Bottom): Error bars SEM; significance calculated using one-way ANOVA. * P ≤ 0.05, **≤0.01, ns = not significant >0.05. Data from four independent biological repeats. N > 50 micronuclei per condition per biological repeat. siSAE:1:SAE2 vs WT P = 0.0163, siSAE:1:SAE2 vs CG P = 0.9986, siSAE:1:SAE2 vs KQ P = 0.8261, siSAE:1:SAE2 vs KR P = 0.0041. ( G ) The percent of metaphase U2OS cells with multipolar spindles in cells expressing siRNA-resistant SAE2 variants after release from 0.332 µM nocodazole, with or without SUMO1 or SUMO2 overexpression. Timeline of the experiment depicted above. Error bars SEM; significance calculated using one-way ANOVA. * P ≤ 0.05, ** P ≤ 0.01, ns = not significant >0.05. Data from three independent biological repeats. N > 50 cells per condition from at least four fields of view per biological repeat. WT vs WT + SUMO1-OE P = 0.0217, WT vs WT + SUMO2-OE P = 0.0007, WT vs KQ + SUMO1-OE P = 0.6593, KQ vs KQ + SUMO1-OE P = 0.0045, KQ vs KQ + SUMO2-OE P = 0.9117. ( H ) Mean percentage of metaphase U2OS cells with multipolar spindles in cells treated with HDAC6 inhibitor, with or without 24 h SUMO1 or SUMO2 overexpression. Timings of the experiment are displayed above. In total, 2.5 µM HDAC6 inhibitor was added 1 hr prior to release from 0.332 µM nocodazole and replaced onto cells for the duration of mitotic release. Error bars SEM; significance calculated using one-way ANOVA, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant >0.05. Data from three independent biological repeats. N > 50 cells per condition from at least four fields of view per biological repeat. Vehicle vs HDAC6i P = 0.0004, Vehicle vs HDAC6i + SUMO1-OE P = 0.0900, Vehicle vs HDAC6i + SUMO2-OE P < 0.0001.

Article Snippet: ML792 , Cambridge Bioscience , HY-108702.

Techniques: Immunoprecipitation, Expressing, Construct, Variant Assay, Over Expression

Journal: The EMBO Journal

Article Title: HDAC6-dependent deacetylation of SAE2 enhances SUMO1 conjugation for mitotic integrity

doi: 10.1038/s44318-025-00532-y

Figure Lengend Snippet: ( A ) Western blot analysis of U2OS expressing FLAG-SAE2r-WT transfected with a GFP-NuMA fragment treated with 0.332 µM nocodazole and 5 µM ML792 ± 5 µM ACY-738 (HDAC6i) for 16 h, alongside FLAG-SAE2r-K164Q expressing cells (5 µM ML792). Mitotic cells were harvested by mitotic shake-off, lysed, and subjected to GFP-trap and western blots probed for SUMO1. The lysates were also precipitated by anti-FLAG beads and probed with antibodies for acK164-SAE2 and SAE2, while inputs were probed for GFP, SUMO1, and GAPDH. Replicated three times in the laboratory. ( B ) Example images of 2, 3, and 4 NuMA structures in metaphase U2OS cells immunostained for NuMA and with Hoechst. White bar indicates 5 micrometers. ( C ) Average percentage of the metaphase cell population with 2 (grey), 3 (blue), 4 (green), and 5+ (purple) NuMA structures in untransfected U2OS cells treated with 2.5 μM HDAC6i (left-hand side of the graph) or cells expressing FLAG-SAE2r-K164Q and treated with 1 μM ML792 (right-hand side of the graph). Cells were also transfected with c-terminal fragments of GFP-NuMA, GFP-NuMA-K1766R, and GFP-SUMO1-NuMA-K1766R. Means are plotted with error bars as SEM for N = 4 independent repeats ( > 50 cells counted per condition) with one-way ANOVA used to assess the statistical significance for the percentage of metaphase cells with 2 NuMA structures, where * P < 0.05 and ns = P > 0.05. Statistical values are - vs HDAC6i P < 0.0001, - vs HDAC6i + GFP-NuMA P < 0.0001, - vs HDAC6i + GFP-NuMA-K1766R P < 0.0001, - vs HDAC6i + GFP-SUMO1-NuMA-K1766R P > 0.9999, HDAC6i + GFP-NuMA-K1766R vs HDAC6i + GFP-SUMO1-NuMA-K1766R P = 0.0009, ML792 vs ML792 + SAE2-WT P < 0.0001, ML792 + SAE2-WT vs ML792 + SAE2-K164Q P < 0.0001, ML792 + SAE2-WT vs ML792 + SAE2-K164Q + GFP-NuMA P = 0.0002, ML792 + SAE2-WT vs ML792 + SAE2-K164Q + GFP-NuMA-K1766R P < 0.0001, ML792 + SAE2-WT vs ML792 + SAE2-K164Q + GFP-SUMO1-NuMA-K1766R P > 0.9999, ML792 + SAE2-K164Q + GFP-NuMA-K1766R vs ML792 + SAE2-K164Q + GFP-SUMO1-NuMA-K1766R P = 0.0008. ( D ) Diagram of NuMA monomer indicating the dominant SUMO1ylation site (purple sphere) at K1766 in the C-terminal ‘self-assembly’ domain (Seo et al, ). Shown below are the GFP-NuMA C-terminal constructs, GFP-NuMA-1708-1982, GFP-NuMA-K1766R-1708-1982, and GFP-SUMO1-NuMA-K1766R-1708-1982 and GFP-SUMO2-NuMA-K1766R-1708-1982 linear fusions. ( E ) Western blot for the GFP-tag showing U2OS transfected with GFP-NuMA constructs. Performed once. ( F ) Representative metaphase cell images of U2OS cells expressing GFP-NuMA, immunostained for α-tubulin, showing bipolar and multipolar (3) spindles. White bar indicates 5 μm. ( G ) Mean percentage of the metaphase cell population with multipolar spindles in U2OS expressing FLAG-SAE2r-K164Q treated with 1 μM ML792 with WT or K1766R C-terminal fragment NuMA variants or SUMO1 fused C-terminal K1766R- NuMA fragment and stained for α-tubulin. N = 9 biological repeats, bars = SEM, and statistical significance was calculated using one-way ANOVA where * P < 0.05, and ns = P > 0.05. Statistical values for - vs SAE2-WT P = 0.1402, ML792 vs ML792 + SAE2-WT P < 0.0001, ML792 + SAE2-WT vs ML792 + SAE2-K164Q + GFP-SUMO1-NuMA-K1766R P > 0.9999, ML792 + SAE2-K164Q vs ML792 + SAE2-K164Q + GFP-NuMA P = 0.0290, ML792 + SAE2-K164Q vs ML792 + SAE2-K164Q + GFP-NuMA-K1766R P = 0.9482, ML792 + SAE2-K164Q vs ML792 + SAE2-K164Q + GFP-SUMO1-NuMA-K1766R P = 0.0004, ML792 + SAE2-K164Q + GFP-NuMA-K1766R vs ML792 + SAE2-K164Q + GFP-SUMO1-NuMA-K1766R P = 0.0042. ( H ) Average percentage of the metaphase cell population with multipolar spindles as assessed by α-tubulin structures in U2OS cells treated with 2.5 μM HDAC6 inhibitor, ACY-738, transfected with GFP-C-terminal fragments, GFP-SUMO1-NuMA-K1766R or GFP-SUMO2-NuMA-K1766R fusion constructs. N = 7 independent biological experiments, bars = SEM, and statistical significance calculated using one-way ANOVA where * P < 0.05 and ns = P > 0.05. Statistical values are – vs HDAC6i P < 0.0001, – vs HDAC6i + GFP-NuMA P = 0.0306, – vs HDAC6i + GFP-NuMA-K1766R P = 0.0010, – vs HDAC6i + GFP-SUMO1-NuMA-K1766R P = 0.9329, – vs HDAC6i + GFP-SUMO2-NuMA-K1766R P = 0.0075.

Article Snippet: ML792 , Cambridge Bioscience , HY-108702.

Techniques: Western Blot, Expressing, Transfection, Construct, Staining