Journal: Leukemia

Article Title: Distinct characteristics of VEXAS-causative UBA1 M41 and recurrent functional non-M41 mutations

doi: 10.1038/s41375-025-02775-4

Figure Lengend Snippet: A Schematic representation of the UBA1 gene, highlighting its functional domains and pinpointing the exact locations of the identified UBA1 variants. The variants were classified into Tier-1 (pathogenic: P), Tier-2 (likely pathogenic: LP) and Tier-3 (variant of uncertain significance: VUS), as depicted in the outer pie chart on the left. UBA1 variants are displayed on top of the gene if the locus occurred recurrently in our screen. Combination of variants detected in the same patients are also shown. Variants detected only as combinations are italicized. M41 and other tier assignments are color-coded. Bold underlined variants were tested for functional significance. B Chart summarizing UBA1 variants and their functional status, indicating the presence or absence of ubiquitylation. Quantification of polyubiquitin levels and mono-ubiquitylated histone H2A/B (Ub-H2A, Ub-H2B) or E2 enzymes (UBE2D3-Ub, UBE2L3-Ub) were normalized within each sample to β-actin levels and scaled to WT transfection. E2 enzyme ubiquitylation was quantified as the ratio of charged form to uncharged form and scaled to WT. Data represent n = 3–6 biological replicates, shown as mean −/+ s.d., significance determined by unpaired t-test with Welch’s correction (*p < 0.05, **p < 0.01).

Article Snippet: Primary antibodies for Poly-ubiquitin (Cell Signaling, 3936S), UBE2D3 (Cell-Signaling, 4330), UBE2L3 (R&D Systems, E2-640), ubiquitylated-H2A (Cell Signaling, 8240), H2A (Cell Signaling, 12349), ubiquitylated-H2B (Cell Signaling, 5546S), H2B (Cell Signaling, 12364), and β-actin (Cell Signaling, 4970) were used at a concentration of 1:1000 and visualized using HRP-conjugated secondary antibodies (anti-rabbit [Cell Signaling, 7074S] or anti-mouse [Cell Signaling, 7076S]) at a concentration of 1:3000.

Techniques: Functional Assay, Variant Assay, Transfection

Journal: Science Advances

Article Title: MYO10 drives genomic instability and inflammation in cancer

doi: 10.1126/sciadv.abg6908

Figure Lengend Snippet: ( A ) A549 control or UbcH7 stably depleted cells were immunostained with anti-UbcH7 and anti–lamin A/C antibodies. Representative images are shown. ( B ) Percentage of cells from (A) and fig. S1C with abnormal nucleus (Nu) including blebs, ruptures, protrusions, etc., were counted. Scoring was not blind. ( C ) Circularity of the nucleus from cells in (A) and fig. S1C. ( D ) A549 control or UbcH7 stably depleted cells were immunostained with anti–lamin B1 antibodies. Representative images are shown. Quantitative results of abnormal nuclear structures ( E ) or the nucleus circularity ( F ) from cells in (D). ( G ) UbcH7-depleted A549 cells were stably transfected with vector control or RNAi-resistant FLAG-UbcH7, and protein expression was examined by Western blot. ( H ) Representative images of the nuclear morphology visualized by anti–lamin A/C antibody from cells generated in (G). Quantitation of abnormal nuclear structures ( I ) or circularity ( J ) from cells in (H). Data represent average and SEM from three independent experiments. Scale bars, 10 μm. ** P < 0.001.

Article Snippet: The following commercially available antibodies were used: anti-MYO10 [Santa Cruz Biotechnology, #sc-166720, or Proteintech, #24565-1-AP, for immunofluorescence staining], anti-UbcH7 (Novus, #NB100-2265), anti–lamin A/C (Santa Cruz Biotechnology, #sc-7292), anti-Nesprin3 (Abcam, #ab74261), anti-ANAPC2 (Santa Cruz Biotechnology, #sc-20984), anti-Flag M2 (Sigma-Aldrich, #F3165), anti-CHK1 (Santa Cruz Biotechnology, #sc-56291), anti-CTSL (Santa Cruz Biotechnology, #sc-32320), anti–tubulin 4A (GeneTex, #GTX112141), anti-synemin (Santa Cruz Biotechnology, #sc-374484), anti-actin (Santa Cruz Biotechnology, #sc-47778), anti-GFP (Novus, #NB100-1770, or Proteintech, #50430-2-AP), anti-Neu (Santa Cruz Biotechnology, #sc-33684), anti–glyceraldehyde phosphate dehydrogenase (Proteintech, #60004), anti-V5 (Cell Signaling Technology, #13202S), anti-p65/RELA (Bethyl Laboratories, #A301-824A), anti-STING (Cell Signaling Technology, #13647S), anti-cGAS (Cell Signaling Technology, #15102S), anti-pSTAT1 (Cell Signaling Technology, #9167S), anti-pSTAT3 (Cell Signaling Technology, #9131S), anti–IL-1β (NCI-monoclonal 3ZD), anti–IL-8 (Santa Cruz Biotechnology, #sc-376750), anti-pTBK1 (Cell Signaling Technology, #5483S), anti-pIRF3 (Cell Signaling Technology, #29047S), and anti–phospho-histone H3 (Ser 10 ) (Millipore, #06-570).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Western Blot, Generated, Quantitation Assay

Journal: Science Advances

Article Title: MYO10 drives genomic instability and inflammation in cancer

doi: 10.1126/sciadv.abg6908

Figure Lengend Snippet: ( A ) Summary of SILAC (top) and RNA sequencing (bottom) data for cytosolic and nuclear structural genes in UbcH7-depleted cells relative to parental A549 cells. Gene families are indicated below. IFs, intermediate filaments; LAPs, lamin-associating proteins; ERM, actin-linkage to membrane. ( B ) A549 control or UbcH7-depleted cells were treated with 320 μM CHX for indicated times, and protein expression was examined by specific antibodies. The band intensity of MYO10 was quantitated and shown above. ( C ) A549 parental and UbcH7-depleted cells were treated with 320 μM CHX with or without 15 μM MG132 for 6 hours, and protein expression was examined. ( D ) Parental and UbcH7-depleted A549 cells were immunoprecipitated (IPed) with rabbit immunoglobulin G (IgG) or rabbit anti-MYO10 antibodies and blotted with indicated antibodies. Short and long exposures for the UbcH7 blot were shown. ( E ) Human embryonic kidney (HEK) 293T cells were transfected with GFP-MYO10 for 48 hours, IPed with the rabbit anti-GFP antibody, and blotted with indicated antibodies. Ten percent of input was also run (bottom). ( F ) Breast cancer HCC1143 and HCC70 cells were transfected with Flag-UbcH7 wild type (WT) or the C86S mutant for 48 hours, and protein expression was examined using specific antibodies.

Article Snippet: The following commercially available antibodies were used: anti-MYO10 [Santa Cruz Biotechnology, #sc-166720, or Proteintech, #24565-1-AP, for immunofluorescence staining], anti-UbcH7 (Novus, #NB100-2265), anti–lamin A/C (Santa Cruz Biotechnology, #sc-7292), anti-Nesprin3 (Abcam, #ab74261), anti-ANAPC2 (Santa Cruz Biotechnology, #sc-20984), anti-Flag M2 (Sigma-Aldrich, #F3165), anti-CHK1 (Santa Cruz Biotechnology, #sc-56291), anti-CTSL (Santa Cruz Biotechnology, #sc-32320), anti–tubulin 4A (GeneTex, #GTX112141), anti-synemin (Santa Cruz Biotechnology, #sc-374484), anti-actin (Santa Cruz Biotechnology, #sc-47778), anti-GFP (Novus, #NB100-1770, or Proteintech, #50430-2-AP), anti-Neu (Santa Cruz Biotechnology, #sc-33684), anti–glyceraldehyde phosphate dehydrogenase (Proteintech, #60004), anti-V5 (Cell Signaling Technology, #13202S), anti-p65/RELA (Bethyl Laboratories, #A301-824A), anti-STING (Cell Signaling Technology, #13647S), anti-cGAS (Cell Signaling Technology, #15102S), anti-pSTAT1 (Cell Signaling Technology, #9167S), anti-pSTAT3 (Cell Signaling Technology, #9131S), anti–IL-1β (NCI-monoclonal 3ZD), anti–IL-8 (Santa Cruz Biotechnology, #sc-376750), anti-pTBK1 (Cell Signaling Technology, #5483S), anti-pIRF3 (Cell Signaling Technology, #29047S), and anti–phospho-histone H3 (Ser 10 ) (Millipore, #06-570).

Techniques: RNA Sequencing Assay, Expressing, Immunoprecipitation, Transfection, Mutagenesis

Journal: Science Advances

Article Title: MYO10 drives genomic instability and inflammation in cancer

doi: 10.1126/sciadv.abg6908

Figure Lengend Snippet: ( A ) U2OS parental, MYO10 +/− , and MYO10 +/− cells stably expressing different levels of GFP-MYO10 (clones #2 to 4). Quantitation of MYO10 protein levels is shown above from three replicates. Quantitation of abnormal nuclear structures ( B ) or cellular circularity ( C ) from cells in (A). ( D ) UbcH7-depleted A549 cells were infected with lentivirus shRNA targeting MYO10 for indicated times, and protein expression was examined. Quantitation of abnormal nuclear structures ( E ) or cellular circularity ( F ) from cells in (D). ( G ) Parental A549 cells were stained with antibodies against MYO10 and lamin A/C. Representative images are shown. Scale bar, 10 μm. ( H ) U2OS parental, MYO10 +/− , and MYO10 +/− cells stably expressing GFP-MYO10 (clone #3) were IPed with MYO10 or lamin A/C and blotted with indicated antibodies. Inputs (5%) from parallel cell samples were run for protein expression. Short and long exposures for MYO10 are provided. ( I ) A549 control or SUN1-depleted cells were IPed with the anti-MYO10 antibody and blotted with indicated antibodies. Data represent average and SEM from three replicates. * P < 0.05. ns, not significant.

Article Snippet: The following commercially available antibodies were used: anti-MYO10 [Santa Cruz Biotechnology, #sc-166720, or Proteintech, #24565-1-AP, for immunofluorescence staining], anti-UbcH7 (Novus, #NB100-2265), anti–lamin A/C (Santa Cruz Biotechnology, #sc-7292), anti-Nesprin3 (Abcam, #ab74261), anti-ANAPC2 (Santa Cruz Biotechnology, #sc-20984), anti-Flag M2 (Sigma-Aldrich, #F3165), anti-CHK1 (Santa Cruz Biotechnology, #sc-56291), anti-CTSL (Santa Cruz Biotechnology, #sc-32320), anti–tubulin 4A (GeneTex, #GTX112141), anti-synemin (Santa Cruz Biotechnology, #sc-374484), anti-actin (Santa Cruz Biotechnology, #sc-47778), anti-GFP (Novus, #NB100-1770, or Proteintech, #50430-2-AP), anti-Neu (Santa Cruz Biotechnology, #sc-33684), anti–glyceraldehyde phosphate dehydrogenase (Proteintech, #60004), anti-V5 (Cell Signaling Technology, #13202S), anti-p65/RELA (Bethyl Laboratories, #A301-824A), anti-STING (Cell Signaling Technology, #13647S), anti-cGAS (Cell Signaling Technology, #15102S), anti-pSTAT1 (Cell Signaling Technology, #9167S), anti-pSTAT3 (Cell Signaling Technology, #9131S), anti–IL-1β (NCI-monoclonal 3ZD), anti–IL-8 (Santa Cruz Biotechnology, #sc-376750), anti-pTBK1 (Cell Signaling Technology, #5483S), anti-pIRF3 (Cell Signaling Technology, #29047S), and anti–phospho-histone H3 (Ser 10 ) (Millipore, #06-570).

Techniques: Stable Transfection, Expressing, Clone Assay, Quantitation Assay, Infection, shRNA, Staining

Journal: Science Advances

Article Title: MYO10 drives genomic instability and inflammation in cancer

doi: 10.1126/sciadv.abg6908

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with GFP, GFP-MYO10, and FLAG–β-TrCP1 or FLAG-Cul1 for 48 hours, IPed with the anti-GFP antibody, and blotted with indicated antibodies. Ten percent of input was run to show protein expression. ( B ) HEK293T cells were transfected with GFP, GFP-MYO10, and FLAG-β–TrCP1–FL (full-length) or FLAG-β–TrCP1–ΔF (F-box deleted) for 48 hours; IPed with the anti-GFP antibody; and blotted with indicated antibodies. Five percent of input was run to show protein expression. ( C ) A549 control or UbcH7-depleted cells were IPed with anti-MYO10 and blotted with antibodies for endogenous proteins. Input (5%) was also run for loading control. ( D ) HEK293T cells were transfected with FLAG–β-TrCP1 or FLAG-β–TrCP1–ΔF for 48 hours and treated with 320 μM CHX for indicated times, and protein expression was examined by specific antibodies. ( E ) The band intensity of MYO10 in (D) was quantitated using the ImageJ software and normalized to that of the 0-hours group. ( F ) A549 cells were transfected with siRNA control or targeting β-TrCP1 for 48 hours and treated with 320 μM CHX for indicated times, and protein expression was examined by specific antibodies. ( G ) Quantitation of MYO10 band intensity in (F) from three replicates. ( H ) HEK293T cells were transfected with FLAG–β-TrCP1 or FLAG-β–TrCP1–ΔF with His-Ub for 48 hours, treated with 320 μM CHX and 15 μM MG132 for 6 hours, lysed cells under denaturing conditions, and proceeded to detect MYO10 ubiquitination as described previously . Ten percent of input was run for loading control. The band intensity of ubiquitinated MYO10 in the IP was quantitated by ImageJ from three blots and presented as average relative intensity (numbers below), which showed a statistical significance ( P = 0.0042) between lanes 2 and 4. Quantitation data are presented as average and SD from at least three experiments. ** P < 0.0001.

Article Snippet: The following commercially available antibodies were used: anti-MYO10 [Santa Cruz Biotechnology, #sc-166720, or Proteintech, #24565-1-AP, for immunofluorescence staining], anti-UbcH7 (Novus, #NB100-2265), anti–lamin A/C (Santa Cruz Biotechnology, #sc-7292), anti-Nesprin3 (Abcam, #ab74261), anti-ANAPC2 (Santa Cruz Biotechnology, #sc-20984), anti-Flag M2 (Sigma-Aldrich, #F3165), anti-CHK1 (Santa Cruz Biotechnology, #sc-56291), anti-CTSL (Santa Cruz Biotechnology, #sc-32320), anti–tubulin 4A (GeneTex, #GTX112141), anti-synemin (Santa Cruz Biotechnology, #sc-374484), anti-actin (Santa Cruz Biotechnology, #sc-47778), anti-GFP (Novus, #NB100-1770, or Proteintech, #50430-2-AP), anti-Neu (Santa Cruz Biotechnology, #sc-33684), anti–glyceraldehyde phosphate dehydrogenase (Proteintech, #60004), anti-V5 (Cell Signaling Technology, #13202S), anti-p65/RELA (Bethyl Laboratories, #A301-824A), anti-STING (Cell Signaling Technology, #13647S), anti-cGAS (Cell Signaling Technology, #15102S), anti-pSTAT1 (Cell Signaling Technology, #9167S), anti-pSTAT3 (Cell Signaling Technology, #9131S), anti–IL-1β (NCI-monoclonal 3ZD), anti–IL-8 (Santa Cruz Biotechnology, #sc-376750), anti-pTBK1 (Cell Signaling Technology, #5483S), anti-pIRF3 (Cell Signaling Technology, #29047S), and anti–phospho-histone H3 (Ser 10 ) (Millipore, #06-570).

Techniques: Transfection, Expressing, Software, Quantitation Assay