Journal: bioRxiv

Article Title: Biological differences in promyelocytic leukemia (PML) proteins between PML-nuclear bodies (PML-NBs) and extranuclear PML bodies (EnPBs) in arsenite-exposed cells

doi: 10.64898/2026.01.25.701640

Figure Lengend Snippet: (A) Immunofluorescence micrographs of U-2OS cells. The cells were exposed to 3 μM As 3+ for 2 or 72 h, or left untreated. After immunostaining for PML and SUMO2/3 antibodies, the cells were counterstained with DAPI (Nuc). Scale bar = 20 μm. An arrow indicates an EnPB. (B) Western blot analyses for PML and SUMO2/3 in U-2OS cells. The cells were treated with either 0.3 or 3 μM As 3+ for 2 or 72 h, or left untreated, and then lyzed with ice-cold RIPA buffer. The lysate was separated into soluble and insoluble fractions by centrifugation. (C) Western blot analyses for PML, SUMO2/3, SUMO1, and ubiquitin in U-2OS cells. The cells were pretreated with 20 μM ML792 (SUMO E1 inhibitor) or 10 μM TAK294 (ubiquitin E1 inhibitor) for 3 h, and were exposed to 3 μM As 3+ for 2 h in the presence of the inhibitor. (D) Immunofluorescence micrographs of PML -/- U-2OS cells. The cells were exposed to 3 μM As 3+ for 2 or 72 h, or left untreated. Scale bar = 20 μm. (E) Western blot analyses for PML and SUMO2/3 in the soluble fractions of wild U-2OS and PML -/- U-2OS cells. Note that SUMOylated proteins were detected in low electromobility region in both cell types. An open arrowhead indicates SUMO monomers. A right curly bracket indicates PML isoforms.

Article Snippet: ML792 (SUMO E1 inhibitor) and TAK294 (ubiquitin E1 inhibitor) were obtained from MedKoo Bioscience (Morrisville, NC) and Selleckchem (Houston, TX), respectively.

Techniques: Immunofluorescence, Immunostaining, Western Blot, Centrifugation, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Biological differences in promyelocytic leukemia (PML) proteins between PML-nuclear bodies (PML-NBs) and extranuclear PML bodies (EnPBs) in arsenite-exposed cells

doi: 10.64898/2026.01.25.701640

Figure Lengend Snippet: (A) Fluorescent micrographs of CHOgPrSWild cells. The cells were pretreated with 20 μM ML792 for 1 h and exposed to 3 μM As 3+ for 2 h, or pretreated with 3 μM As 3+ for 1 h and exposed to ML792 for 1 or 18 h. Scale bar = 10 μm. (B) Western blot analyses for PML, SUMO2/3, GFP, and RFP (mCherry) in the RIPA-soluble and -insoluble fractions of CHOgPrSWild cells. The cells were pretreated with 3 μM As 3+ for 2 h or 20 μM ML792 for 1 h, and then exposed to ML792 for 1 h or As 3+ for 2 h, respectively. Closed and open arrowheads indicate GFP-tagged PML-VI and SUMO2/3 monomers, respectively. An arrow indicates an mCherry-tagged SUMO2.

Article Snippet: ML792 (SUMO E1 inhibitor) and TAK294 (ubiquitin E1 inhibitor) were obtained from MedKoo Bioscience (Morrisville, NC) and Selleckchem (Houston, TX), respectively.

Techniques: Western Blot

Journal: Molecular Cell

Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

doi: 10.1016/j.molcel.2020.11.029

Figure Lengend Snippet: RFWD3 simulates ubiquitylation of proteins on ssDNA (A) Fpg bacterial glycosylase was crosslinked to either double-stranded (pFpg) or single-stranded DNA (pFpg ssDNA ) and added to SPRTN-depleted non-licensing egg extracts. DPC pull-down under stringent conditions was performed at the indicated time points, and samples were blotted against crosslinked Fpg. Slow mobility bands represent ubiquitylated Fpg species (see B). (B) pFpg ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and samples were blotted against Fpg as in (A). (C) pFpg ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (also depleted of SPRTN) for the indicated time points and samples processed as in (A). (D) Generation of an AP site on ssDNA (pAP ssDNA ) to induce HMCES crosslinking. (E) pAP ssDNA was incubated in SPRTN-depleted non-licensing extracts, and ubiquitin E1 inhibitor was added where indicated. Plasmids were recovered, and proteins were blotted against HMCES. The black dot indicates sumoylated HMCES (see F). (F) pAP ssDNA was incubated in mock- or RFWD3-depleted non-licensing extracts (depleted of SPRTN), and ubiquitin E1 inhibitor or SUMO E1 inhibitor was added where indicated. Plasmids were recovered and analyzed as in (D). (G) Model illustrating the role of RFWD3 in gap-filling DNA synthesis (see ).

Article Snippet: SUMO E1 inhibitor (ML-792; Medkoo Biosciences) was supplemented to egg extracts at the final concentration of 50 μM.

Techniques: Incubation, Ubiquitin Proteomics, DNA Synthesis

Journal: Molecular Cell

Article Title: The ubiquitin ligase RFWD3 is required for translesion DNA synthesis

doi: 10.1016/j.molcel.2020.11.029

Figure Lengend Snippet:

Article Snippet: SUMO E1 inhibitor (ML-792; Medkoo Biosciences) was supplemented to egg extracts at the final concentration of 50 μM.

Techniques: Ubiquitin Proteomics, Recombinant, Mutagenesis, Staining, Magnetic Beads, Transfection, Sequencing, Software

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