Journal: Advanced Science

Article Title: Targeting Nupr1‐Mediated p53 Mitochondrial Sequestration Alleviates Aortic Dissection by Restoring the Gadd45b‐Dependent Survival Program

doi: 10.1002/advs.78143

Figure Lengend Snippet: Genetic and Pharmacological Inhibition of Nupr1 Attenuates Ang II Induced Mitochondrial Apoptosis in VSMCs. (A) Cell ability was evaluated by CCK8 assay ( n = 8 per group). (B) Lactate production of indicated groups ( n = 6 per group). (C) Representative images of TEM in VSMCs from the indicated groups. (D) Quantification of mitochondrial size in VSMCs from indicated groups ( n = 6 per group). (E) Quantification of circularity index in VSMC from indicated groups ( n = 6 per group). (F) Real‐time monitoring of oxygen consumption rate (OCR) in indicated groups detected by O2k. (G–I) Quantification of ATP‐coupled respiration, mitochondrial basal respiration, and maximal respiration from indicated groups ( n = 6 per group). (J) Representative images of calcein and Annexin V fluorescence results showing mPTP opening and apoptosis in indicated groups. Stronger green fluorescence represents a lower degree of mPTP opening, while diminished fluorescence indicates increased mPTP opening. Stronger red fluorescence represents a higher apoptosis rate. (K, L) Quantification of fluorescence intensity, including Annexin V and Calcein ( n = 6 per group). (M) Flow cytometry analysis of Annexin and PI staining of apoptosis from indicated groups. (N) Quantification of early apoptosis, late apoptosis, and total apoptosis by flow cytometry ( n = 3 per group). (O) Quantification of maximal respiration from indicated groups ( n = 6 per group). (P) Real‐time monitoring of OCR in indicated groups detected by O2k. (Q, R) Quantification of ATP‐coupled respiration and mitochondrial basal respiration from indicated groups ( n = 6 per group). (S) Flow cytometry analysis of Annexin and PI staining of apoptosis from indicated groups. (T) Quantification of early apoptosis, late apoptosis, and total apoptosis by flow cytometry ( n = 3 per group).

Article Snippet: According to mPTP test kit instructions (C2009S, Beyotime), 200 μL of Calcein AM staining solution and fluorescence quenching working solution were added to each well after the respective cell treatments.

Techniques: Inhibition, CCK-8 Assay, Real-time Monitoring, Fluorescence, Flow Cytometry, Analysis, Staining

Journal: Advanced Science

Article Title: Targeting Nupr1‐Mediated p53 Mitochondrial Sequestration Alleviates Aortic Dissection by Restoring the Gadd45b‐Dependent Survival Program

doi: 10.1002/advs.78143

Figure Lengend Snippet: Gadd45b is Indispensable for the Protective Effects of Nupr1 Deficiency Against Mitochondrial Apoptosis. (A) Enrichment of KEGG pathways. (B) GSEA analysis in VSMCs treated with Ang II stimulation and Nupr1 deficiency. (C) Chordal diagram of GO pathways related with cell fate and the contributing DEPs. (D) Stem plot of DEPs in the apoptosis pathway from the indicated groups. (E) RT‐qPCR analysis of mRNA levels of candidate genes in VSMCs. Data were presented as relative fold change to controls ( n = 6 per group). (F) Expressions of Gadd45b in VSMCs measured by Western blot and its quantification from indicated groups measured by Western blot ( n = 5 per group). (G, H) Representative images of ROS staining in indicated groups. Semi‐quantitative analysis of mean fluorescence intensity in indicated groups ( n = 6 per group). (I) Representative images of calcein and Annexin V fluorescence results showing mPTP opening in indicated groups. (J) Quantification of fluorescence intensity, including Annexin V and mPTP ( n = 3 per group). (K) Co‐immunoprecipitation and Western blot assays of Myc‐Nupr1 and Gadd45b. (L) Co‐immunoprecipitation assays of Myc‐Gadd45b and Nupr1.

Article Snippet: According to mPTP test kit instructions (C2009S, Beyotime), 200 μL of Calcein AM staining solution and fluorescence quenching working solution were added to each well after the respective cell treatments.

Techniques: Analysis, Quantitative RT-PCR, Western Blot, Staining, Fluorescence, Co-Immunoprecipitation Assay, Significance Assay

Journal: MedComm

Article Title: The PIEZO1/CISD1/IP3R Mechanotransduction Axis Drives Macrophage Mitochondrial Dysfunction and Inflammatory Activation in Pulmonary Fibrosis

doi: 10.1002/mco2.71080

Figure Lengend Snippet: Increased mechanical stiffness induces MPTP opening and mitochondrial damage in macrophages. (A) MH‐S cells were incubated with different stiffness matrix gels for 24 h. Mitochondrial membrane potential was labeled using the mitochondrial membrane potential probe JC‐1 and detected by flow cytometry, n = 6 biological replicates. (B). MH‐S cells were incubated with different stiffness matrix gels for 24 h. Mitochondrial reactive oxygen species content in cells was measured by using the mitoSOX probe and detected by flow cytometry, n = 5 biological replicates. (C). MH‐S cells were incubated with different stiffness matrix gels for 24 h. Calcium ion concentration in cytoplasm was detected using flow cytometry, n = 5 biological replicates. (D). MH‐S cells were incubated with different stiffness matrix gels for 24 h. MPTP activity of macrophages after matrix gel culture was assayed using the MPTP kit, scale bar = 100 µm, n = 6 biological replicates, stronger green fluorescence corresponds to reduced MPTP opening and preserved mitochondrial integrity, while weaker fluorescence indicates elevated MPTP opening and impaired mitochondrial structural integrity. (E). MH‐S cells were incubated with different stiffness matrix gels for 24 h. Expression of mitochondrial membrane permeability transition pore‐associated genes ANT1 and CypD detected by qPCR, n = 5 biological replicates. (F). MH‐S cells were incubated with different stiffness matrix gels for 24 h. Expression of ANT1 and CypD was detected by WB, n = 5 biological replicates. (G). MH‐S cells were incubated with different stiffness matrix gels for 24 h. Expression of CD163 and iNOS was detected by WB, n = 6 biological replicates.

Article Snippet: MPTP activity was detected using the MPTP assay kit (Beyotime Biotech, Shanghai, China).

Techniques: Incubation, Membrane, Labeling, Flow Cytometry, Concentration Assay, Activity Assay, Fluorescence, Expressing, Permeability

Journal: MedComm

Article Title: The PIEZO1/CISD1/IP3R Mechanotransduction Axis Drives Macrophage Mitochondrial Dysfunction and Inflammatory Activation in Pulmonary Fibrosis

doi: 10.1002/mco2.71080

Figure Lengend Snippet: Knockdown of CISD1 attenuates mechanical stiffness‐mediated macrophage MPTP opening and mitochondrial damage. (A). MH‐S cells with knockdown of CISD1 were cultured on different stiffness matrix gels, and cytosolic calcium ions were detected by flow cytometry, n = 5 biological replicates. (B). MH‐S cells with knockdown of CISD1 were cultured on different stiffness matrix gels, and MPTP activity was detected by flow cytometry, n = 5 biological replicates. (C). MH‐S cells with knockdown of CISD1 were cultured on different stiffness matrix gels, mitochondrial reactive oxygen species content was detected by flow cytometry, n = 5 biological replicates. (D and E) MH‐S cells with knockdown of CISD1 were cultured on different stiffness matrix gels, mitochondrial membrane potential was detected by immunofluorescence microscope, n = 3 biological replicates, scale bar = 20 µm. (F and G). MH‐S cells with knockdown of CISD1 were cultured on different stiffness matrix gels, and MPTP‐related proteins and inflammation‐related proteins were detected by WB, n = 4 biological replicates.

Article Snippet: MPTP activity was detected using the MPTP assay kit (Beyotime Biotech, Shanghai, China).

Techniques: Knockdown, Cell Culture, Flow Cytometry, Activity Assay, Membrane, Immunofluorescence, Microscopy

Journal: MedComm

Article Title: The PIEZO1/CISD1/IP3R Mechanotransduction Axis Drives Macrophage Mitochondrial Dysfunction and Inflammatory Activation in Pulmonary Fibrosis

doi: 10.1002/mco2.71080

Figure Lengend Snippet: Matrix stiffness upregulates CISD1 via PIEZO1 to mediate mitochondrial damage. (A) MH‐S cells were treated with 2.5 µM or 5 µM Yoda1 for 24 h. WB assay was performed to detect the expression of MPTP‐related proteins, n = 6 biological replicates. (B) MH‐S cells were treated with 5 µM Yoda1 for 24 h, mitochondrial reactive oxygen species content was detected by flow cytometry, n = 6 biological replicates. (C) MH‐S cells were treated with 5 µM Yoda1 for 24 h, mitochondrial membrane potential was detected by immunofluorescence microscopy, n = 6 biological replicates, scale bar = 100 µm. (D) MH‐S cells were treated with 5 µM Yoda1 for 24 h, MPTP opening was detected by immunofluorescence microscopy, n = 6 biological replicates, scale bar = 100 µm. (E) MH‐S cells were cultured with matrix gels of different stiffness and simultaneously treated with PIEZO1 inhibitor GsMTx4, WB assay was performed to detect the expression of MPTP‐related proteins, n = 4 biological replicates. (F) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with PIEZO1 inhibitor GsMTx4, and MPTP activity was detected by flow cytometry, n = 3 biological replicates. (G) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with PIEZO1 inhibitor GsMTx4, mitochondrial reactive oxygen species content was detected by flow cytometry, n = 5 biological replicates. (H) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with PIEZO1 inhibitor GsMTx4, mitochondrial membrane potential was detected by immunofluorescence microscopy, n = 4 biological replicates, scale bar = 20 µm.

Article Snippet: MPTP activity was detected using the MPTP assay kit (Beyotime Biotech, Shanghai, China).

Techniques: Expressing, Flow Cytometry, Membrane, Immunofluorescence, Microscopy, Cell Culture, Activity Assay

Journal: MedComm

Article Title: The PIEZO1/CISD1/IP3R Mechanotransduction Axis Drives Macrophage Mitochondrial Dysfunction and Inflammatory Activation in Pulmonary Fibrosis

doi: 10.1002/mco2.71080

Figure Lengend Snippet: Matrix stiffness‐mediated mitochondrial damage depends on IP3R activity. (A) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with IP3R inhibitor 2‐APB, and cytosolic calcium ions were detected by flow cytometry, n = 3 biological replicates. (B) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with IP3R inhibitor 2‐APB, and MPTP activity was detected by flow cytometry, n = 3 biological replicates. (C) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with IP3R inhibitor 2‐APB, mitochondrial reactive oxygen species content was detected by flow cytometry, n = 3 biological replicates. (D) MH‐S cells were cultured with different stiffness matrix gels and simultaneously treated with IP3R inhibitor 2‐APB, mitochondrial membrane potential was detected by immunofluorescence microscopy, n = 3 biological replicates, scale bar = 20 µm.

Article Snippet: MPTP activity was detected using the MPTP assay kit (Beyotime Biotech, Shanghai, China).

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Membrane, Immunofluorescence, Microscopy

Journal: Advanced Science

Article Title: H4K12 Lactylation Impairs Dopaminergic Neuron Energy Metabolism via the HIF‐1α/NDUFS1 Pathway in Parkinson's Disease

doi: 10.1002/advs.78220

Figure Lengend Snippet: H4K12la regulates HIF‐1α expression in PD models, and inhibition of lactylation by 2‐DG ameliorates related pathologies. (A) Western blotting analysis of H4K12la and HIF‐1α protein levels in SN4741 cells treated with 0.5 mM MPP + , NaLa, Lac, or NaCl for 24 h (N = 3). (B) Representative immunofluorescence images of SN4741 cells showing H4K12la (green) and HIF‐1α (red) expression in the nucleus under the indicated treatments. DAPI (blue) stains nuclei. Scale bar, 100 µm. Insets show 2 × magnifications of the boxed regions. (C) Quantitative analysis of the relative fluorescence intensity of H4K12la and HIF‐1α. (D,E) Western blotting analysis and quantification of H4K12la and HIF‐1α protein levels in the (D) striatum and (E) substantia nigra of MPTP‐induced PD mice (n = 3). (F, G) RT‐qPCR analysis of HIF‐1α mRNA levels in the (F) striatum and (G) substantia nigra of MPTP‐induced PD mice (n = 3). (H,I) Cells transfected with H4‐WT or H4‐K12R were treated with MPP + . H4K12la and HIF‐1α protein levels were assessed by Western blotting (N = 3). (J) Intracellular lactate levels in MPP + ‐treated SN4741 cells with or without 2‐DG treatment (N = 3). (K) Western blotting analysis of H4K12la and HIF‐1α protein levels in MPP + ‐treated SN4741 cells with or without 2‐DG co‐treatment (N = 3). (L) Representative immunofluorescence images of SN4741 cells showing that 2‐DG co‐treatment reduces MPP + ‐treated elevations of H4K12la (green) and HIF‐1α (red). Scale bar, 100 µm. (M) Corresponding quantification of fluorescence intensity for H4K12la and HIF‐1α. (N,O) Lactate levels in the (N) substantia nigra and (O) striatum homogenates of MPTP‐induced PD mice with or without 2‐DG treatment (n = 3). (P,Q) Western blotting analysis and quantification of H4K12la and HIF‐1α protein levels in the (P) striatum and (Q) substantia nigra of MPTP‐induced PD mice with or without 2‐DG treatment (n = 3). (R) Representative immunofluorescence images of midbrain sections from MPTP‐induced PD mice with or without 2‐DG treatment, showing TH (red) and H4K12la (green) expression. Scale bar, 200 µm (n = 3). Data are presented as mean ± SEM. ns, not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001. Statistical significance was determined by one‐way ANOVA or Student's t‐test as appropriate. Lac, lactate; NaLa, sodium lactate; NaCl, sodium chloride; 2‐DG, 2‐deoxy‐D‐glucose. “n” refers to the number of mice per group. “N” refers to the number of biologically independent experiments.

Article Snippet: Mice in the 2‐DG + MPTP group were pretreated with 2‐DG (400 mg/kg, MCE, #HY‐13966) via intraperitoneal injection once daily for 3 days prior to MPTP administration.

Techniques: Expressing, Inhibition, Western Blot, Analysis, Immunofluorescence, Fluorescence, Quantitative RT-PCR, Transfection

Journal: Advanced Science

Article Title: H4K12 Lactylation Impairs Dopaminergic Neuron Energy Metabolism via the HIF‐1α/NDUFS1 Pathway in Parkinson's Disease

doi: 10.1002/advs.78220

Figure Lengend Snippet: 2‐DG ameliorates energy metabolism deficits, provides neuroprotection, and improves motor function in PD models by regulating H4K12la. (A,B) Measurement of (A) the ATP levels and (B) NAD + /NADH ratio in SN4741 cells treated with MPP + , with or without 2‐DG (N = 3). (C) Experimental schedule. (D–G) Measurement of the ATP levels and NAD + /NADH ratio in the (D,E) striatum and (F,G) substantia nigra of MPTP‐induced mice treated with or without 2‐DG (n = 3). (H) Representative immunohistochemical images and quantitative analysis of TH expression in the midbrain of mice treated with MPTP, with or without 2‐DG (n = 3). Scale bar, 200 µm. (I,J) Western blotting analysis and quantification of ATPB protein levels in the (I) striatum and (J) substantia nigra of mice treated with MPTP, with or without 2‐DG (n = 3). (K) RT‐qPCR analysis of TH mRNA levels in the substantia nigra of mice treated with MPTP, with or without 2‐DG (n = 3). (L–N) Behavioral assessments of MPTP‐induced mice with or without 2‐DG treatment: (L) time in the pole climbing test. (M) open‐field test showing representative images of mouse movement trajectories and total distance traveled. (N) Scores in the grasping test. n = 7 per group. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001. Statistical significance was determined by one‐way ANOVA or Student's t‐test as appropriate. ATPB, ATP synthase subunit beta. “n” refers to the number of mice per group. “N” refers to the number of biologically independent experiments.

Article Snippet: Mice in the 2‐DG + MPTP group were pretreated with 2‐DG (400 mg/kg, MCE, #HY‐13966) via intraperitoneal injection once daily for 3 days prior to MPTP administration.

Techniques: Immunohistochemical staining, Analysis, Expressing, Western Blot, Quantitative RT-PCR