Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 1 Identification of MYSM1 as a regulator of DOX-induced cardiotoxicity. A-B. Heatmaps showing the expression profiles of JAMMs family genes in RNA-seq from mouse cardiac tissues (GSE224157, A) and human cardiac tissues (GSE133054, B, HF = heart failure, NHF = non-heart failure). C. HL-1 cells were transfected with overexpressing plasmids of JAMMs family (e.g. oeMYSM1) or empty vector (EV) plasmid before doxorubicin (DOX) treatment (1 µM, 24 h). Cell viability was measured by CCK8 assay (*, vs. EV; #, vs. EV + DOX; # P < 0.05, *** P < 0.001, n = 6). D. Comprehensive analysis combining RNA-seq datasets and functional screening of JAMMs family for identification of MYSM1 as a regulator in DOX-induced cardiotoxicity. E-F. mRNA levels of Mysm1 in DOX-treated HL-1 cells (E) or mouse cardiac tissues (F) were detected by RT-qPCR (n = 8). G-H. Protein levels of MYSM1 in DOX-treated HL-1 cells (G) or mouse cardiac tissues (H) were assessed by western blotting (n = 3). I. Representative immunofluorescence staining for MYSM1 (red), cardiomyocytes marker α-actinin (green), fibroblasts marker vimentin (green), or macrophages marker F4/80 (green) in heart sections from DOX-treated mice. Slides were counterstained with DAPI (blue). Yellow in merged images denotes regions of colocalization. J. mRNA levels of MYSM1 in cardiomyocytes (CMs), fibroblasts (Fbs), macrophages (Macs), and endothelial cells (ECs) were detected by RT-qPCR under DOX injury (n = 8). (ns = no significance, ** P < 0.01, *** P < 0.001)

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: Expressing, RNA Sequencing, Transfection, Plasmid Preparation, CCK-8 Assay, Functional Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Marker

Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 2 Knockdown of MYSM1 alleviates DOX-induced cardiotoxicity. A. Workflow depicting intraperitoneal injection of DOX at a dosage of 15 mg/kg/ week (three times per week for 2 weeks) in Mysm1−/+mice. Cardiac function evaluation was performed four weeks after the first DOX injection. B-D. Levels of serum lactic dehydrogenase (LDH, B), creatine kinase-MB (CK-MB, C), and cardiac troponin T (cTnT, D) following DOX treatment (n = 7). E. Representative left ventricular M-mode echocardiographic images. F-G. Relative levels of ejection fraction (EF%, F) and fractional shortening (FS%, G) in mice (n = 7). H. Representative whole heart images. I. Representative images of hematoxylin and eosin (H&E) of whole heart sections. J-K. Ratio of heart weight to tibia length (HW/TL, J) and heart weight to body weight (HW/BW, K) (n = 7). L-M. Cardiomyocyte cross-sectional area was assessed by wheat germ agglutinin (WGA, L) and statistical results (M, n = 7). N. Representative images of longitudinal H&E sections of cardiac tissues. O-P. Representative images of Sirius Red (O) and quantification analysis (P, n = 7). Q-R. Detection of apoptotic DNA fragmentation in cardiac tissues by TUNEL staining (Q) and corresponding statistical results (R, n = 7). (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: Knockdown, Injection, TUNEL Assay, Staining

Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 3 Cardiomyocyte specific knockdown of MYSM1 protects against DOX-induced cardiotoxicity. A AAV9-cTNT-shMYSM1(total dose 2 × 1011 vg/mouse) was administered to mice via tail vein injection. Four weeks later, mice were intraperitoneally injected with DOX at a dosage of 15 mg/kg/week (three times per week for 2 weeks). Cardiac performance was evaluated four weeks after the first DOX injection. B-D. Levels of serum LDH (B), CK-MB (C), and cTnT (D) following DOX treatment (n = 7). E. Representative left ventricular M-mode echocardiographic images. F-G. Relative levels of EF% (F) and FS% (G) in mice (n = 7). H. Representative whole heart images. I. Representative images of H&E of whole heart sections. J-K. Ratio of HW/TL (J) and HW/BW (K) in mice (n = 7). L-M. Cardiomyocyte cross-sectional area was assessed by WGA (L), and statistical results (M, n = 7). N. Representative images of longitudinal H&E sections of cardiac tissues. O-P. Representative images of Sirius Red (O) and quantification analysis (P, n = 7). Q-R. Detection of apoptotic DNA frag mentation in cardiac tissues by TUNEL staining (Q) and corresponding statistical results (R, n = 7). (ns = no significance, * P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: Knockdown, Injection, TUNEL Assay, Staining

Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 4 MYSM1 mediates DOX-induced cardiomyocyte injury in vitro. A-D Construction of MYSM1-KD HL-1 cells before DOX treatment. (A) Cell viability of each group (n = 6). (B) Bar graph showing the levels of LDH (n = 6). (C, D) The effect of MYSM1 knockdown on DOX-induced apoptosis in HL-1 cells was determined by propidium iodide (PI) staining. Representative images for PI staining are shown in D, with quantitative analysis for PI-positive cells in C (n = 3). E-H. Overexpression of MYSM1 by transfecting HL-1 cells with oeMYSM1 before DOX treatment. (E) Cell viability of each group (n = 6). (F) Bar graph showing the levels of LDH (n = 6). (G-H) The effect of oeMYSM1 on DOX-induced apoptosis in HL-1 cells was determined by PI staining. Representa tive images for PI staining are shown in H, with quantitative analysis for PI-positive cells in G (n = 3). I-L MYSM1 was knocked down by siRNA transfection (siMYSM1) in neonatal rat primary cardiomyocytes (NRPCs) before DOX treatment. (I) Cell viability of each group (n = 6). (J) Bar graph showing the levels of LDH (n = 6). (K-L) The effect of siMYSM1 on DOX-induced apoptosis in NRPCs was determined by PI staining. Representative images for PI staining are shown in L, with quantitative analysis for PI-positive cells in K (n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: In Vitro, Knockdown, Staining, Over Expression, Transfection

Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 5 MYSM1 interacts with TRIM21 to maintain the stability of TRIM21 protein. (A) Schematic diagram of the experiment for screening MYSM1 sub strates by Co-immunoprecipitation (Co-IP) combined with liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis. HL-1 cells were transfected with Flag-MYSM1 or Flag- empty vector (EV) plasmid before DOX injury. Cells were harvested using lysis buffer and incubated with anti-Flag-beads for Co-IP. The binding proteins were extracted, digested to peptide, and then subjected to LC-MS/MS analysis. (B) Verification of the Co-IP of Flag-MYSM1 by immunoblotting. (C) Two-dimensional plot with X-axis showing protein scores and Y-axis showing protein sequence coverage. (D) The potential substrate protein with the highest-ranking score for MYSM1. E-F. Co-IP of MYSM1 and TRIM21 in cardiac tissues (E) and HL-1 cells (F) treated with DOX. Endogenous TRIM21 was immunoprecipitated by anti-TRIM21 antibody. G. Co-IP of MYSM1 and TRIM21 in NIH/3T3 cells transfected with Flag- MYSM1 and Myc-TRIM21. Exogenous Flag-MYSM1 was immunoprecipitated by anti-Flag antibody. H. Immunofluorescence staining was performed to identify the colocalization of Flag-MYSM1 (green) and TRIM21(red) in DOX treated HL-1 cells. Yellow in merged images denotes regions of colocalization. I. NIH/3T3 cells were transfected with oeMYSM1 (1–2 µg) and analyzed by western blotting for MYSM1 and TRIM21 protein; GAPDH was used as a loading control. J. RT-qPCR analysis of mRNA levels of Mysm1 and Trim21 in NIH/3T3 cells transfected with increasing amounts of oeMYSM1 (n = 8). K-L. NIH/3T3 cells were transfected with Flag-MYSM1 or EV plasmid and subjected to cycloheximide (CHX, 25 µg/mL). MYSM1 and TRIM21 protein levels were detected by western blotting (K), with densitometric quantification of TRIM21 (L, ns = no significance; *, vs. Vector-6; #, vs. Vector-9; **P < 0.01, ## P < 0.01, n = 3). M. NIH/3T3 cells were co-transfected with Flag-MYSM1, Myc-TRIM21, HA-Ub, HA-K48, and HA-K63, followed by treatment with 10 µM MG132 for 4 h before collection. Cell lysates were treated with anti-Myc antibody, and ubiquitinated TRIM21 was detected by western blotting

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Lysis, Incubation, Binding Assay, Western Blot, Sequencing, Immunofluorescence, Staining, Control, Quantitative RT-PCR

Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 6 MYSM1 binds to TRIM21 via the SWIRM domain and deubiquitinates TRIM21 in an MPN domain-dependent manner. (A) Schematic of MYSM1 and its deletion mutants, including MYSM1(∆SANT), MYSM1(∆SWIRM), and MYSM1(∆MPN). (B) NIH/3T3 cells were co-transfected with Myc-TRIM21, Flag- MYSM1-WT or ∆MPN, ∆SWIRM and ∆SANT mutants, followed by treatment with 10 µM MG132 for 4 h before collection. Cell lysates were treated with anti-Flag antibody to identify the binding region of MYSM1 that regulates deubiquitination of TRIM21. C-D. NIH/3T3 cells were transfected with Flag- MYSM1-WT or MPN mutants, subjected to CHX (25 µg/mL), and then analyzed by western blotting for MYSM1, TRIM21 and GAPDH (C), densitometric quantification of TRIM21 (D, ns = no significance; *, vs. WT-6; #, vs. WT-9; ** P < 0.01, ## P < 0.01, n = 3). E. NIH/3T3 cells were co-transfected with Myc- TRIM21, HA-Ub, Flag-MYSM1-WT or ∆MPN mutants, followed by treatment with 10 µM MG132 for 4 h before collection. Cell lysates were treated with anti-Myc antibody to identify the active region of MYSM1 that regulates deubiquitination of TRIM21. F. Schematic representation of the mechanism of MYSM1’s deubiquitination of TRIM21

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: Transfection, Binding Assay, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: Deubiquitinase MYSM1 promotes doxorubicin-induced cardiotoxicity by mediating TRIM21-ferroptosis axis in cardiomyocytes.

doi: 10.1186/s12964-024-01955-6

Figure Lengend Snippet: Fig. 7 MYSM1 mediates ferroptosis in DOX-induced cardiotoxicity. A-D. MYSM1-KD HL-1 cells were treated with DOX (1 µM) for 24 h. Commercial assay kits were used to detect the level of Fe2+ (A, n = 6), representative malondialdehyde (MDA) content (B, n = 6), glutathione (GSH) levels (C, n = 6), and super oxide dismutase (SOD) activities (D, n = 6) in the cells. E-H. HL-1 cells were transfected with oeMYSM1 plasmid and then treated with DOX for 24 h. Com mercial assay kits were used to detect the level of Fe2+ (E, n = 6), representative MDA content (F, n = 6), GSH levels (G, n = 6), and SOD activities (H, n = 6) in the cells. I. DHE fluorescence imaging of ROS in MYSM1-KD or MYSM1-overexpression HL-1 cells treated with DOX. J. The mitochondrial membrane potential in MYSM1-KD or MYSM1-overexpression HL-1 cells treated with DOX were measured by JC-1 staining. K-L. The protein levels of nuclear factor (erythroid-derived 2)‐like2 (Nrf2) in nuclear and cytoplasmic were measured in MYSM1-KD or MYSM1-overexpression HL-1 cells treated with DOX using western blotting. M-N. MYSM1-KD or MYSM1-overexpression HL-1 cells were treated with the DOX for 24 h. The protein levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) were measured by western blotting (n = 3). O-R. HL-1 cells were transfected with oeMYSM1 or EV plasmid and treated with Fer-1 (5 µM) for 2 h before DOX treatment. Cell viability measured by CCK8 assay (O, n = 6). Levels of supernatant LDH (P, n = 6). Representative images of Hoechst and PI double fluorescent staining (Q) and statistical results (R, n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: Then the membranes were blocked with 3% BSA for 2 h at room temperature; following this, they were incubated overnight at 4 °C with the following primary antibodies: MYSM1 (abs136708, Absin, 1:1000), TRIM21 (12108-1-AP, Proteintech, 1:1000), γ-H2AX (ab81299, Abcam, 1:1000), SLC7A11 (26864-1-AP, Proteintech, 1:1000), GPX4 (67763-1-lg, Proteintech, 1:1000), Nrf2 (12721, CST, 1:1000), Histone H3 (68345-1-Ig, Proteintech, 1:1000), TUBULIN (2128, CST, 1:1000), and GAPDH (5174, CST, 1:1000).

Techniques: Transfection, Plasmid Preparation, Fluorescence, Imaging, Over Expression, Membrane, Staining, Derivative Assay, Western Blot, CCK-8 Assay

Journal: Scientific Reports

Article Title: SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination

doi: 10.1038/srep17735

Figure Lengend Snippet: ( A ) Schematic representation of murine Specc1l gene indicating insertion of genetrap vectors in ES cell clones DTM096 (intron 1) and RRH048 (intron 15). ( B–D) Heterozygous Specc1l DTM096 embryos stained for lacZ , representing Specc1l expression, from E8.5 to E10.5. NE = neuroectoderm, NF = neural folds, PA1 = 1 st pharyngeal arch. ( E–P) Co-immunostaining of SPECC1L with NCC markers AP2A and SOX10 in E8.5 neural folds (NF; E–J ) and in E9.5 cranial sections ( K–P ). SPECC1L staining is broadly observed in the E8.5 neural folds ( E,H ; arrows), including in cells marked by AP2A (F,G; arrows) and SOX10 ( I,J ; arrows). At E9.5, SPECC1L strongly stains migrating CNCCs ( K,N ; arrows) marked by AP2A ( L,M ; arrows) and SOX10 ( O,P ; arrows).

Article Snippet: Antibodies against β-catenin (rabbit; 1:1000; Santa Cruz, Dallas TX) (mouse; 1:1000; Cell Signaling Technology, Danvers, MA), Myosin IIb (1:1000; Sigma-Aldrich, St. Louis, MO), E-cadherin (1:1000; Abcam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, CO), SOX10 (1:1000; Aviva Systems Biology, San Diego, CA), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA), KI67 (1:1000; Cell Signaling Technology, Danvers MA), cleaved Caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA), and β-actin (1:2500; Sigma-Aldrich, St. Louis, MO) were used as described.

Techniques: Clone Assay, Staining, Expressing, Immunostaining

Journal: Scientific Reports

Article Title: SPECC1L deficiency results in increased adherens junction stability and reduced cranial neural crest cell delamination

doi: 10.1038/srep17735

Figure Lengend Snippet: ( A,B’) E9.5 WT ( A ) embryo with migratory cranial neural crest cells (CNCCs) marked with Wnt1 -Cre ( A ’). In contrast, Specc1l mutant embryo shows open neural folds ( B ), arrow) with CNCCs that have still not migrated ( B ’, arrow). ( C,D’) Brightfield images ( C,D ) and CNCC marker DLX2 immuno-stain ( C’,D’ ) of E10.5 WT ( C,C’ ) and Specc1l mutant ( D,D’ ) embryos. In E10.5 WT embryo, DLX2 positive CNCCs populate the branchial arches ( C’ , arrowhead), while in the mutant significant staining remains in open neural folds ( D’ , arrow) with some staining in the 1 st pharyngeal arch ( D’ , arrowhead), indicating poor CNCC delamination and migration. E-R) Sections from WT and Specc1l mutant embryos at E8.5 ( E–L ) and at E9.5 ( M–R ) were stained with NCC markers SOX10 ( E,F,M,N ), AP2A ( G,H,O,P ), and DLX2 ( I,J,Q,R ). At E8.5, NCC staining is observed in the neural folds (NF) of WT and mutant sections. Co-staining of SOX10 and β-catenin in E8.5 WT ( K ) and mutant ( L ) shows increased β-catenin staining at cell boundaries in the neural folds. At E9.5, migratory CNCC staining is observed in WT ( M,O,Q ), while in mutants undelaminated CNCCs stain the open neural folds ( N,P,R ). ( S–Z) Analysis of AJ markers in vivo in WT and Specc1l DTM096/RRH048 mutant E9.5 embryo coronal sections. The approximate plane of section is indicated in the top-right corner. Increased F-actin ( S,T ) and Myosin IIb ( U,V ) staining is observed in mutant tissue sections. Similarly to in vitro results in , expanded β-catenin ( W,X ) and E-cadherin ( Y,Z ) membrane staining is observed in vivo in mutant embryos. ( AA-BB) Electron micrograph of WT embryo section looking at apico-basal cell boundary shows a distinct electron-dense region indicating adherens junction ( AA , arrowhead). In contrast, the entire apico-basal boundary appears electron-dense in Specc1l mutant embryo section ( BB , arrowheads), suggesting increased density and dispersion of adherens junctions.

Article Snippet: Antibodies against β-catenin (rabbit; 1:1000; Santa Cruz, Dallas TX) (mouse; 1:1000; Cell Signaling Technology, Danvers, MA), Myosin IIb (1:1000; Sigma-Aldrich, St. Louis, MO), E-cadherin (1:1000; Abcam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, CO), SOX10 (1:1000; Aviva Systems Biology, San Diego, CA), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA), KI67 (1:1000; Cell Signaling Technology, Danvers MA), cleaved Caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA), and β-actin (1:2500; Sigma-Aldrich, St. Louis, MO) were used as described.

Techniques: Mutagenesis, Marker, Immunostaining, Staining, Migration, In Vivo, In Vitro, Membrane, Dispersion