Journal: Advanced Science

Article Title: Mitigating Doxorubicin‐Induced Cardiotoxicity and Enhancing Anti‐Tumor Efficacy with a Metformin‐Integrated Self‐Assembled Nanomedicine

doi: 10.1002/advs.202415227

Figure Lengend Snippet: Design and mechanism of PMDDH. A) Schematic representation of metformin enhancing the cytotoxic effect of Dox on 4T1 cells and mitigating its cardiotoxicity on H9c2 cells. B) Synthesis of PMD, PMDD, and PMDDH. C) Illustration of the targeted delivery, enhanced therapeutic efficacy, and reduced cardiotoxicity of PMDDH in vivo.

Article Snippet: Meanwhile, the 4T1 cells were collected and resuspended in a Cell Staining Buffer (Elabscience, Wuhan, China).

Techniques: Drug discovery, In Vivo

Journal: Advanced Science

Article Title: Mitigating Doxorubicin‐Induced Cardiotoxicity and Enhancing Anti‐Tumor Efficacy with a Metformin‐Integrated Self‐Assembled Nanomedicine

doi: 10.1002/advs.202415227

Figure Lengend Snippet: A) Visualization of cellular uptake of Dox, free FAM‐DNA, and PMDDH by CLSM. Scale bar = 20 µm. B,C) Flow cytometry analysis and corresponding quantitative results of PMDDH uptake in 4T1 cells ( n = 3). D) Visualization of CD44 expression (green) and PMDDH uptake (red) in 4T1, MDA‐MB‐231, and MCF‐7 cells. Scale bar = 20 µm. E) Dose‐dependent cytotoxicity of Dox, PMDH, and PMDDH in 4T1 cells ( n = 5). F) IC 50 values of Dox and PMDDH in 4T1 cells ( n = 5). G) Crystal violet staining of treated 4T1 cells showing cell density and distribution changes. H) CRT expression (red) in 4T1 cells post‐treatment. Scale bar = 50 µm. I) Flow cytometry analysis of CRT expression and J) corresponding quantitative results ( n = 3). K) HMGB1 levels in the supernatant of 4T1 cells following treatment ( n = 3). L) ATP release from 4T1 cells after 24 h of different treatments ( n = 3). Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: Meanwhile, the 4T1 cells were collected and resuspended in a Cell Staining Buffer (Elabscience, Wuhan, China).

Techniques: Flow Cytometry, Expressing, Staining

Journal: Advanced Science

Article Title: Mitigating Doxorubicin‐Induced Cardiotoxicity and Enhancing Anti‐Tumor Efficacy with a Metformin‐Integrated Self‐Assembled Nanomedicine

doi: 10.1002/advs.202415227

Figure Lengend Snippet: A) Schematic illustration of cGAS‐STING and AMPK signaling activation in 4T1 cells. B) Western blot analysis of p‐IRF3 and p‐STING levels in 4T1 cells after different treatments. RT‐qPCR analysis of cytokine and chemokine expression: C) IFN‐β, D) CXCL10, E) OAS1, F) ISG15, and G) TNF‐α in 4T1 cells. H) Western blot results showing p‐AMPK and p‐mTOR levels in 4T1 cells post‐treatment. I) Flow cytometry analysis and J) immunofluorescence imaging of PD‐L1 expression in 4T1 cells following various treatments. Scale bar = 20 µm. Data are presented as mean ± SD ( n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Meanwhile, the 4T1 cells were collected and resuspended in a Cell Staining Buffer (Elabscience, Wuhan, China).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Imaging

Journal: Advanced Science

Article Title: Mitigating Doxorubicin‐Induced Cardiotoxicity and Enhancing Anti‐Tumor Efficacy with a Metformin‐Integrated Self‐Assembled Nanomedicine

doi: 10.1002/advs.202415227

Figure Lengend Snippet: A) Pharmacokinetics of free Dox and PMDDH in rats, with inset showing the drug‐time curve of the distribution phase ( n = 3). B) In vivo distribution of free Cy5‐DNA compared to Cy5‐labeled PMDDH. C) Ex vivo fluorescence imaging of tumors and major organs in tumor‐bearing mice following intravenous administration of free Cy5‐DNA and PMDDH. D) Schematic of treatment regimen for 4T1 tumor‐bearing mice. E) Tumor growth curves after various treatments over 16 days ( n = 5). F) Tumor inhibitory rates and G) tumor weights at day 16 ( n = 5). H) Histological evaluation of tumor tissues using H&E staining, Ki67 immunohistochemistry, and TUNEL immunofluorescence. Scale bars = 100 µm for H&E staining; 200 µm for Ki67 immunohistochemistry and TUNEL immunofluorescence. Data are presented as mean ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Meanwhile, the 4T1 cells were collected and resuspended in a Cell Staining Buffer (Elabscience, Wuhan, China).

Techniques: Drug discovery, In Vivo, Labeling, Ex Vivo, Fluorescence, Imaging, Staining, Immunohistochemistry, TUNEL Assay, Immunofluorescence

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency results in excessive activation of NLRP3 inflammsome in macrophages. Two groups of macrophages, i.e., peritoneal macrophages from conditional SHP2 knockout (cSHP2-KO) and wild-type (WT) mice a–c , or PMA-differentiated THP-1 cells with shRNA-Ctrl or shRNA-SHP2 lentivirus d , f , were primed with 100 ng ml −1 LPS for 3 h, and then stimulated with ATP (5 mM, 1 h), MSU (500 µg ml −1 , 2 h) or Nigericin (10 µM, 2 h), respectively. a , b , d , e Enzyme-linked immunosorbent assay (ELISA) of IL-1β and IL-18 in culture supernatants. ND represents not detectable. c , f Immunoblot analysis of cell lysates. g ELISA of IL-1β in supernatants of THP-1-derived macrophages left untreated or treated with SHP2 inhibitor PHPS1 (10 μM) or NSC87877 (10 μM) for 1 h, followed by LPS treatment and ATP, MSU or Nigericin stimulation. h Immunoblot analysis of proteins immunoprecipitated with anti-ASC from lysates of ATP-treated SHP2 knockdown THP-1-derived macrophages. i Immunoblot analysis of ASC in cross-linked pellets (upper panels) and cell lysates (lower panels) from ATP-treated SHP2 knockdown THP-1-derived macrophages. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons. Data are representative of three independent experiments (mean and SEM of three independent samples in a , b , d , e , g )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Knock-Out, shRNA, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Immunoprecipitation, Knockdown

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency in macrophages aggravates murine peritonitis model. a – c Eight-week-old female cSHP2-KO and WT mice were killed 12 h after Alum injection and peritoneal cavities were washed with PBS. a Flow cytometry analysis of peritoneal exudate cells (PECs) 12 h after Alum injection in mice. b ELISA of IL-1β level in the lavage fluid 8 h after Alum injection in mice. c Immunoblot analysis (left panel) and flow cytometry analysis (right panel) of caspase-1 activation in PECs 12 h after Alum injection in mice. Data are representative of three independent experiments (mean and SEM of 10 mice per group), * P < 0.05 by Student’s t -test

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Activation Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 deficiency leads to mitochondrial dysfunction and excessive NLRP3 inflammasome activation. a , b Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining a or mitochondrial ROS by MitoSOX staining b in peritoneal macrophages from conditional SHP2 knockout (cSHP2-KO) and wild-type (WT) mice, and left untreated or treated with ATP (5 mM) for indicated times. c Quantitative real-time PCR analysis of mtDNA released from peritoneal macrophages from cSHP2-KO and WT mice and left unstimulated (medium) or primed with LPS (100 ng ml −1 ) for 3 h and stimulated with ATP (5 mM, 1 h) and Nigericin (10 µM, 2 h). d ELISA of IL-1β in supernatants of peritoneal macrophages from cSHP2-KO and WT mice, which were primed with LPS (100 ng ml −1 ) for 3 h, and left untreated or treated with Ac-YVAD-cmk (30 μM), NAC (5 mM), or E-64d (20 μM) for 1 h, followed by stimulation of ATP (5 mM) for 1 h. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons; NS represents no significance. Data are presented as mean ± SEM of three independent experiments in a – d

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Flow Cytometry, Membrane, Staining, Knock-Out, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 translocates into mitochondrial matrix during NLRP3 inflammasome activation. a Immunofluorescence analysis of SHP2 and mitochondria from bone marrow-derived macrophages with untreated (medium) or ATP (5 mM, 15 min), or MSU (500 µg ml −1 , 2 h) or Nigericin (10 µM, 2 h) treatment. Scale bar, 5 µm. b Immunoblot analysis of mitochondrial and cytosolic components of THP-1-derived macrophages treated with ATP (5 mM) for indicated times. c Immunoblot analysis of SHP2 location in mitochondria from THP-1-derived macrophages. Cells were treated with 5 mM ATP for 30 min, then mitochondria were isolated and incubated with 40 μM proteinase K for 30 min. Tom20 in mitochondrial outer membrane (MOM) and Tim23 in mitochondrial inner membrane (MIM) were used as controls, respectively. d Immunoblot analysis of SHP2 expression in submitochondrial fractions from THP-1-derived macrophages treated with ATP (5 mM, 30 min). Tom20, COX IV, and HSP60 were used to represent MOM, MIM, and mitochondrial matrix protein, respectively. e Immunofluorescence analysis of SHP2 and Tom20 from bone marrow-derived macrophages with untreated (medium) or ATP (5 mM, 30 min) by structured-illumination microscopy (SIM). Scale bar, 5 µm. Data are representative of three independent experiments

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Immunofluorescence, Derivative Assay, Western Blot, Isolation, Incubation, Membrane, Expressing, Microscopy

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 interacts with ANT1 during NLRP3 inflammasome activation. a Sliver staining of GST pull-down proteins using GST-SHP2 fusion protein. The proteins interacting with GST-SHP2 were identified by mass spectrometry. b Immunofluorescence analysis of SHP2 and ANT1 from bone marrow-derived macrophages with untreated (medium) or ATP (5 mM, 15 min) treatment. Scale bar, 5 µm. c Immunoblot analysis of reciprocal co-immunoprecipitation (Co-IP) from THP-1-derived macrophages treated with ATP (5 mM) for indicated times. d Immunoblot analysis of Co-IP from mitochondrial and cytosolic components in THP-1-derived macrophages treated with ATP for 15 min. e Immunoblot analysis of reciprocal Co-IP from HEK293T cells overexpressing HA-tagged SHP2 and myc-tagged ANT1. Data are representative of three independent experiments

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Staining, Mass Spectrometry, Immunofluorescence, Derivative Assay, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: Tom20/Tom40 and Tim23 complex are necessary for SHP2 translocation to mitochondrial matrix. a Predication of mitochondrial target sequence in SHP2 by PSORT II. b Immunoblot analysis of GFP localization in submitochondrial fractions from HEK293T cells which were transfected with GFP or RRWFH-GFP plasmid. c Immunoblot analysis of SHP2 localization in submitochondrial fractions from HEK293T cells which were transfected with SHP2-HA or SHP2-mut-HA (mitochondrial target sequence mutation, RRWFH mutated to AAWFH) plasmid followed by ATP treatment (5 mM, 30 min). d Immunofluorescence analysis mitochondrial localization of GFP-tagged RRWFH motif in HEK293T cells. Scale bar, 10 µm. e Immunofluorescence analysis mitochondrial localization of SHP2-HA or SHP2-mut-HA plasmid in HEK293T cells. Scale bar, 10 µm. f Co-immunoprecipitation (Co-IP) analysis of the interaction of SHP2 and ANT1 in HEK293T cells, which were transfected with ANT1-myc and SHP2-HA or SHP2-mut-HA. g Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining in HEK293T cells which were transfected with SHP2-HA or SHP2-mut-HA plasmid followed by cccp treatment (20 µM, 1 h). h Immunoblot analysis of SHP2 in mitochondria after Tom20 or Tom40, or Tom70 sliencing. Endogenous Toms were separately knocked down by its corresponding shRNAs in THP-1 cells followed by ATP treatment (5 mM, 30 min), then mitochondria were isolated and incubated with 40 μM proteinase K (Pro K) for 30 min. Tom20 in mitochondrial outer membrane (MOM) and Tim23 in mitochondrial inner membrane (MIM) were used as control, respectively. i Immunoblot analysis of SHP2 expression in submitochondrial fractions from THP-1-derived macrophages treated with ATP (5 mM, 30 min) after Tim22 or Tim23 silencing. * P < 0.05 by Student’s t -test, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in g )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Translocation Assay, Sequencing, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunofluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Flow Cytometry, Membrane, Staining, Isolation, Incubation, Control, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 inhibits NLRP3 inflammasome activation in an ANT1-dependent manner. a , b SHP2 knockdown, ANT1 knockdown and SHP2-ANT1 double knockdown THP-1-derived macrophages were primed with 100 ng ml −1 LPS for 3 h, followed by ATP (5 mM, 1 h), MSU (500 µg ml −1 , 2 h), or Nigericin (10 µM, 2 h) stimulation, respectively. a ELISA of IL-1β in the supernatant. b Quantitative real-time PCR analysis of mtDNA. c ELISA of IL-1β in the culture supernatant from ANT1 knockdown THP-1-derived macrophages and left untreated or treated with NSC87877 (10 μM) or PHPS1 (10 μM) for 1 h, followed by ATP or Nigericin stimulation. d – f LPS-primed SHP2 knockdown THP-1-derived macrophages were treated with CATR (5 mM) or BA (50 µM) for 1 h, followed by ATP or Nigericin stimulation. d Immunoblot analysis of Co-IP from THP-1-derived macrophages treated with CATR or BA. e ELISA of IL-1β in the culture supernatant. f Immunoblot analysis of cell lysates from THP-1-derived macrophages treated with CATR or BA. * P < 0.05, ** P < 0.01, one-way ANOVA for multiple comparisons, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in a – c , e )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay, Knockdown, Derivative Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: SHP2 dephosphorylation of ANT1 at Tyr 191 is essential for mitochondrial homeostasis. a Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining of HEK293T cells overexpressing Vector, SHP2-HA, SHP2-D61A-HA, or SHP2-C459S-HA plasmid and left untreated (medium) or treated with cccp (20 µM, 1 h). b , c HEK293T cells were transfected with pro-caspase-1, ASC, NLRP3, and SHP2-HA, SHP2-D61A-HA, or SHP2-C459S-HA plasmid, respectively, followed by ATP (5 mM, 1 h) treatment. b Flow cytometry analysis of caspase-1 activation. c Immunoblot analysis of caspase-1 activation. d Flow cytometry analysis of mitochondrial membrane potential by JC-1 staining of HEK293T cells overexpressing Vector, ANT1-myc, ANT1-Y191F-myc, or ANT1-Y195F-myc plasmid and left untreated (medium) or treated with cccp (20 µM, 1 h). e , f HEK293T cells were transfected with pro-caspase-1, ASC, NLRP3, and ANT1-myc, ANT1-Y191F-myc or ANT1-Y195F-myc plasmid respectively followed by ATP (5 mM, 1 h) treatment. e Flow cytometry analysis of caspase-1 activation. f Immunoblot analysis of caspase-1 activation. * P < 0.05, one-way ANOVA for multiple comparisons, NS represents no significance. Data are representative of three independent experiments (mean and SEM of three independent samples in a , b , d , e )

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: De-Phosphorylation Assay, Flow Cytometry, Membrane, Staining, Plasmid Preparation, Transfection, Activation Assay, Western Blot

Journal: Nature Communications

Article Title: Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1-dependent mitochondrial homeostasis

doi: 10.1038/s41467-017-02351-0

Figure Lengend Snippet: The graphic illustration of the mechanism of SHP2 regulating NLRP3 inflammasome activation. Stimulated by Signal 2 activators (e.g., ATP, MSU, and Nigericin), SHP2 is recruited to mitochondria through its RRWFH motif. With the help of Tom20/Tom40 and Tim23 complex, SHP2 translocates into the mitochondrial matrix and dephosphorylates ANT1 at Tyr 191. This serves as a key mechanism controlling mitochondrial homeostasis, preventing leakage of mitochondrial DNA (mtDNA) and overproduction of reactive oxygen species (ROS), which results in negative regulation of NLRP3 inflammasome activation

Article Snippet: pET21b-caspase-1-His (Plasmid 11809), pCI-ASC-HA (Plasmid 41553), pGEX-4T1 SHP2 WT (Plasmid 8322), and pCMV-SHP2 (Plasmid 8381) were purchased from Addgene.

Techniques: Activation Assay

Journal: Cell reports

Article Title: Breast cancer cell mesenchymal transition and metastasis directed by DAP5/eIF3d-mediated selective mRNA translation

doi: 10.1016/j.celrep.2023.112646

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Murine: 4T1 , ATCC , Cat# CRL-3407/RRID:CVCL_GR31.

Techniques: Recombinant, Staining, Protease Inhibitor, Virus, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, MTT Assay, Software, Imaging