Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Fibroblast growth factor-inducible 14 accelerates pulmonary fibrosis by inducing fibroblast senescence in mice

doi: 10.1007/s00018-026-06161-w

Figure Lengend Snippet: Activation of Fn14 inhibits mitophagy in fibroblasts. A , primary fibroblasts were treated with rTWEAK (100 ng/mL) for 48 h. Cell samples were harvested after 48 h. Volcano plot showing the changes of fibroblast genes (fold change ≥ 2). B , KEGG enrichment analysis for differentially expressed genes in Control vs. rTWEAK-treated primary fibroblasts. C , Mitochondrial membrane potential was measured by JC-1 staining. D , the expression of Mfn1 , Opa1 , and Dnm1 mRNA in primary fibroblasts was detected by Real-time PCR ( n = 3). E-J , The protein levels of L-OPA1, MFN1, FUNDC1, PINK1, and LC3 were determined by Western blot analysis ( n = 3). K , Immunofluorescence and confocal microscopy show the colocalization of Mito Tracker (green) and LAMP1 (red) in rTWEAK-treated primary fibroblasts, scale bar = 50 μm. * P < 0.05 and ** P < 0.01

Article Snippet: Anti-FUNDC1 monoclonal antibody , CST , 49,240 , 1: 2000.

Techniques: Activation Assay, Control, Membrane, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Confocal Microscopy

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Construction of In Vitro Model and Observation of Mitochondrial Autophagy and Expression Changes of c-FLIP, JNK, and FUNDC1 (A and B) Optical microscopy of cardiac microvascular endothelial cell (CMEC) morphology and CCK-8 assay for cell proliferation after 12 hours of different lipopolysaccharide (LPS) concentrations. (C and D) Optical microscopy of CMECs morphology and CCK-8 assay for cell proliferation at various times after 10 μg/mL LPS treatment. (E and F) c-FLIP expression at various times after 10 μg/mL LPS treatment. (G) Transmission electron microscopy (×12,000) of CMECs microstructure to assess mitochondrial damage and autophagy (representative images). (H to L) Western blot of mitochondrial autophagy-related proteins. (M-Q) Expression of c-FLIP, total c-Jun N-terminal kinase (t-JNK), phosphorylated JNK (p-JNK), total Fun14 domain-containing protein 1 (t-FUNDC1), phosphorylated FUNDC1 (p-FUNDC1) proteins, and FUNDC1 mRNA. Note: n = 3/group; 3 technical replicates; mean ± SEM; Student's t -test (N-Q), analysis of variance with Tukey’s post hoc test (B, D, F, and I to L). ∗ P < 0.05 vs Ctrl (control) group, ∗∗ P < 0.01 vs Ctrl group, ∗∗∗ P < 0.001 vs Ctrl group. Abbreviations as in .

Article Snippet: The membranes were then incubated overnight at 4 °C with the following primary antibodies: GAPDH (Xianzhi Biotechnology Co, Ltd AB-P-R 001, 1:1000), COXIV (Wuhan Sanying Biotechnology Co, Ltd, 11242-1-AP, 1:1000), c-FLIP (Affinity Biosciences, DF7010, 1:1000), total FUNDC1 (t-FUNDC1) (Cell Signaling Technology, 49240, 1:1000), phosphorylated FUNDC1 (p-FUNDC1) (Affinity Biosciences, AF0001, 1:1000), total JNK (t-JNK) (Affinity Biosciences, AF6318, 1:1000), p-JNK (Affinity Biosciences, AF3318, 1:1000), IL-6 (Affinity Biosciences, DF6087, 1:1000), IL-8 (Wuhan Sanying Biotechnology Co, Ltd, 27095-1-AP, 1:1000), TNFα (Wuhan Sanying Biotechnology Co, Ltd, 26405-1-AP, 1:1000), MMP9 (Wuhan Sanying Biotechnology Co, Ltd, 27306-1-AP, 1:500), LC3 (Wuhan Sanying Biotechnology Co, Ltd, 14600-1-AP, 1:2500), Tomm20 (Wuhan Sanying Biotechnology Co, Ltd, 11802-1-AP, 1:8000), p62 (Affinity Biosciences, AF5384, 1:1000), cyt C (Wuhan Sanying Biotechnology Co, Ltd, 66264-1-Ig, 1:20000), and caspase-3 (Wuhan Sanying Biotechnology Co, Ltd, 19677-1-AP, 1:500).

Techniques: In Vitro, Expressing, Microscopy, CCK-8 Assay, Transmission Assay, Electron Microscopy, Western Blot, Control

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Changes in Mitochondrial Autophagy Activity at Different Stages of LPS-Induced Injury and the Regulatory Role of c-FLIP (A) Electron microscopy of mitochondrial structure and autophagy in CMECs 12 hours post–LPS-induced injury. Yellow arrows indicate damage to mitochondrial structure; green arrows indicate autophagosomes or autolysosomes. (B to F) Western blot analysis of the expression of proteins related to mitochondrial autophagy in CMECs 12 hours after LPS-induced injury. (G-L) Western blot analysis of the expression of mitochondrial autophagy-related proteins in CMECs 36 hours post–LPS-induced injury. (M and N) Immunofluorescence used to detect the fusion of mitochondria and lysosomes. Green fluorescence stains mitochondria, red fluorescence stains lysosomes, blue fluorescence stains nuclei, and orange-yellow fluorescence indicates the fusion of mitochondria and lysosomes, representing mitochondrial autophagy. Note: n = 3/group; 3 technical replicates; mean ± SEM; analysis of variance with Tukey’s post hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; siRNA-FUNDC1 was applied to silence the FUNDC1 gene, thereby reducing or preventing the production of FUNDC1 protein. ∗ P < 0.05 vs Ctrl group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The membranes were then incubated overnight at 4 °C with the following primary antibodies: GAPDH (Xianzhi Biotechnology Co, Ltd AB-P-R 001, 1:1000), COXIV (Wuhan Sanying Biotechnology Co, Ltd, 11242-1-AP, 1:1000), c-FLIP (Affinity Biosciences, DF7010, 1:1000), total FUNDC1 (t-FUNDC1) (Cell Signaling Technology, 49240, 1:1000), phosphorylated FUNDC1 (p-FUNDC1) (Affinity Biosciences, AF0001, 1:1000), total JNK (t-JNK) (Affinity Biosciences, AF6318, 1:1000), p-JNK (Affinity Biosciences, AF3318, 1:1000), IL-6 (Affinity Biosciences, DF6087, 1:1000), IL-8 (Wuhan Sanying Biotechnology Co, Ltd, 27095-1-AP, 1:1000), TNFα (Wuhan Sanying Biotechnology Co, Ltd, 26405-1-AP, 1:1000), MMP9 (Wuhan Sanying Biotechnology Co, Ltd, 27306-1-AP, 1:500), LC3 (Wuhan Sanying Biotechnology Co, Ltd, 14600-1-AP, 1:2500), Tomm20 (Wuhan Sanying Biotechnology Co, Ltd, 11802-1-AP, 1:8000), p62 (Affinity Biosciences, AF5384, 1:1000), cyt C (Wuhan Sanying Biotechnology Co, Ltd, 66264-1-Ig, 1:20000), and caspase-3 (Wuhan Sanying Biotechnology Co, Ltd, 19677-1-AP, 1:500).

Techniques: Activity Assay, Electron Microscopy, Western Blot, Expressing, Immunofluorescence, Fluorescence

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Mitochondrial Function Changes in the Late Stage of LPS-Induced Injury and the Protective Role of c-FLIP (A and B) Measurement of mitochondrial Ca 2+ levels using the cell-permeant Ca 2+ fluorescent probe Rhod-2 AM, quantified by flow cytometry to reflect Ca 2+ levels inside mitochondria. (C) Quantification of mitochondrial reactive oxygen species (ROS) using the DCFH-DA probe. (D) Measurement of mitochondrial adenosine triphosphate (ATP) content by assessing absorbance. (E and F) Assessment of the mitochondrial permeability transition pore (mPTP) state using the Calcein AM fluorescent probe, with CoCl 2 quenching fluorescence in non-mitochondrial regions; changes in mitochondrial fluorescence intensity are analyzed via flow cytometry, where a decrease in fluorescence indicates mPTP opening. (G and H) Analysis of changes in mitochondrial membrane potential (ΔΨm) using the JC-1 fluorescent probe, with flow cytometry used to analyze the ratio of red to green fluorescence for quantitative assessment. Note: n = 3/group; 3 technical replicates; mean ± SEM; analysis of variance with Tukey’s post-hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; siRNA-FUNDC1 targeted to silence the FUNDC1 gene, thus reducing or inhibiting the production of FUNDC1 protein. ∗ P < 0.05 vs Ctrl group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The membranes were then incubated overnight at 4 °C with the following primary antibodies: GAPDH (Xianzhi Biotechnology Co, Ltd AB-P-R 001, 1:1000), COXIV (Wuhan Sanying Biotechnology Co, Ltd, 11242-1-AP, 1:1000), c-FLIP (Affinity Biosciences, DF7010, 1:1000), total FUNDC1 (t-FUNDC1) (Cell Signaling Technology, 49240, 1:1000), phosphorylated FUNDC1 (p-FUNDC1) (Affinity Biosciences, AF0001, 1:1000), total JNK (t-JNK) (Affinity Biosciences, AF6318, 1:1000), p-JNK (Affinity Biosciences, AF3318, 1:1000), IL-6 (Affinity Biosciences, DF6087, 1:1000), IL-8 (Wuhan Sanying Biotechnology Co, Ltd, 27095-1-AP, 1:1000), TNFα (Wuhan Sanying Biotechnology Co, Ltd, 26405-1-AP, 1:1000), MMP9 (Wuhan Sanying Biotechnology Co, Ltd, 27306-1-AP, 1:500), LC3 (Wuhan Sanying Biotechnology Co, Ltd, 14600-1-AP, 1:2500), Tomm20 (Wuhan Sanying Biotechnology Co, Ltd, 11802-1-AP, 1:8000), p62 (Affinity Biosciences, AF5384, 1:1000), cyt C (Wuhan Sanying Biotechnology Co, Ltd, 66264-1-Ig, 1:20000), and caspase-3 (Wuhan Sanying Biotechnology Co, Ltd, 19677-1-AP, 1:500).

Techniques: Flow Cytometry, Permeability, Fluorescence, Membrane

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Apoptosis and Functional Changes of CMECs in the Late Stage of LPS-Induced Injury (A and B) Optical microscopy observations of CMEC morphology and CCK-8 assay to assess cell proliferation. (C-F) Western blot analysis of changes in the expression of proteins related to the mitochondrial apoptosis pathway. (G and H) Immunofluorescence staining for caspase-9 expression on CMECs. Red fluorescence stains the specific marker CD34 on CMEC surfaces; green fluorescence stains caspase-9; blue fluorescence stains nuclei. (I and J) Flow cytometric analysis to detect apoptosis rates. (K and L) Transwell assays to assess the chemotactic motility of endothelial cells. (M): TER assay to evaluate endothelial barrier function. Note: n = 3/group; 3 technical replicates; mean ± SEM; analysis of variance with Tukey’s post hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; siRNA-FUNDC1 targeted to silence the FUNDC1 gene, thus reducing or preventing the production of FUNDC1 protein. ∗ P < 0.05 vs Ctrl group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The membranes were then incubated overnight at 4 °C with the following primary antibodies: GAPDH (Xianzhi Biotechnology Co, Ltd AB-P-R 001, 1:1000), COXIV (Wuhan Sanying Biotechnology Co, Ltd, 11242-1-AP, 1:1000), c-FLIP (Affinity Biosciences, DF7010, 1:1000), total FUNDC1 (t-FUNDC1) (Cell Signaling Technology, 49240, 1:1000), phosphorylated FUNDC1 (p-FUNDC1) (Affinity Biosciences, AF0001, 1:1000), total JNK (t-JNK) (Affinity Biosciences, AF6318, 1:1000), p-JNK (Affinity Biosciences, AF3318, 1:1000), IL-6 (Affinity Biosciences, DF6087, 1:1000), IL-8 (Wuhan Sanying Biotechnology Co, Ltd, 27095-1-AP, 1:1000), TNFα (Wuhan Sanying Biotechnology Co, Ltd, 26405-1-AP, 1:1000), MMP9 (Wuhan Sanying Biotechnology Co, Ltd, 27306-1-AP, 1:500), LC3 (Wuhan Sanying Biotechnology Co, Ltd, 14600-1-AP, 1:2500), Tomm20 (Wuhan Sanying Biotechnology Co, Ltd, 11802-1-AP, 1:8000), p62 (Affinity Biosciences, AF5384, 1:1000), cyt C (Wuhan Sanying Biotechnology Co, Ltd, 66264-1-Ig, 1:20000), and caspase-3 (Wuhan Sanying Biotechnology Co, Ltd, 19677-1-AP, 1:500).

Techniques: Functional Assay, Microscopy, CCK-8 Assay, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Marker

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: In the Late Stage of LPS-Induced Injury, JNK Participates in the Regulation of FUNDC1-Mediated Mitophagy by c-FLIP and its Potential Mechanisms (A to G) FUNDC1 mRNA expression and Western blot analysis of mitochondrial autophagy-related proteins. (H and I) Immunofluorescence measures the fusion between mitochondria and lysosomes; mitochondria are stained green, lysosomes red, nuclei blue, and merged mitochondria and lysosomes orange-yellow, indicative of mitochondrial autophagy. (J-M) Co-IP using anti–p-JNK or anti–t-FUNDC1 antibodies followed by immunoblotting with anti–t-FUNDC1 or anti–p-JNK antibodies to assess the interaction between proteins, with IgG serving as a negative control. (N and O) Immunofluorescence staining for observing the colocalization of p-JNK and t-FUNDC1, utilizing confocal microscopy to depict their interactions; p-JNK is stained green, t-FUNDC1 red, nuclei blue, and the colocalization of p-JNK and t-FUNDC1 orange, representing their interactions. Note: n = 3/group; 3 technical replicates (Co-IP done twice); mean ± SEM; analysis of variance with Tukey’s post hoc test. Ad-c-FLIP was used to infect CMECs to overexpress the c-FLIP protein; SP600125 (SP) is a JNK inhibitor, and Ani is a JNK activator. ∗ P < 0.05 vs control group, # P < 0.05 vs LPS + Ad-NC CMECs group, @ P < 0.05 vs LPS + Ad-c-FLIP CMECs group. Abbreviations as in , , , and .

Article Snippet: The membranes were then incubated overnight at 4 °C with the following primary antibodies: GAPDH (Xianzhi Biotechnology Co, Ltd AB-P-R 001, 1:1000), COXIV (Wuhan Sanying Biotechnology Co, Ltd, 11242-1-AP, 1:1000), c-FLIP (Affinity Biosciences, DF7010, 1:1000), total FUNDC1 (t-FUNDC1) (Cell Signaling Technology, 49240, 1:1000), phosphorylated FUNDC1 (p-FUNDC1) (Affinity Biosciences, AF0001, 1:1000), total JNK (t-JNK) (Affinity Biosciences, AF6318, 1:1000), p-JNK (Affinity Biosciences, AF3318, 1:1000), IL-6 (Affinity Biosciences, DF6087, 1:1000), IL-8 (Wuhan Sanying Biotechnology Co, Ltd, 27095-1-AP, 1:1000), TNFα (Wuhan Sanying Biotechnology Co, Ltd, 26405-1-AP, 1:1000), MMP9 (Wuhan Sanying Biotechnology Co, Ltd, 27306-1-AP, 1:500), LC3 (Wuhan Sanying Biotechnology Co, Ltd, 14600-1-AP, 1:2500), Tomm20 (Wuhan Sanying Biotechnology Co, Ltd, 11802-1-AP, 1:8000), p62 (Affinity Biosciences, AF5384, 1:1000), cyt C (Wuhan Sanying Biotechnology Co, Ltd, 66264-1-Ig, 1:20000), and caspase-3 (Wuhan Sanying Biotechnology Co, Ltd, 19677-1-AP, 1:500).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Negative Control, Confocal Microscopy, Control

Journal: JACC: Basic to Translational Science

Article Title: c-FLIP Protects Cardiac Microcirculation in Sepsis-Induced Myocardial Dysfunction Via FUNDC1-Mediated Regulation of Mitochondrial Autophagy

doi: 10.1016/j.jacbts.2025.02.016

Figure Lengend Snippet: Protective Mechanism of c-FLIP in SIMD c-FLIP maintains myocardial microcirculation homeostasis by differentially regulating mitochondrial autophagy, whose aberration can lead to SIMD. Specifically, in the early stages, c-FLIP relies on FUNDC1 to modulate mitochondrial autophagy, with a deficiency in c-FLIP leading to enhanced mitochondrial autophagic activity. In the later stages, c-FLIP in the myocardial microcirculation regulates FUNDC1-mediated mitochondrial autophagy via the JNK pathway; a deficiency in c-FLIP results in abnormalities in this signaling pathway, causing suppressed mitochondrial autophagy activity. Changes in mitochondrial autophagy at any stage disrupt mitochondrial homeostasis, impair myocardial microcirculation, and ultimately trigger sepsis-induced myocardial dysfunction. Abbreviations as in and .

Article Snippet: The membranes were then incubated overnight at 4 °C with the following primary antibodies: GAPDH (Xianzhi Biotechnology Co, Ltd AB-P-R 001, 1:1000), COXIV (Wuhan Sanying Biotechnology Co, Ltd, 11242-1-AP, 1:1000), c-FLIP (Affinity Biosciences, DF7010, 1:1000), total FUNDC1 (t-FUNDC1) (Cell Signaling Technology, 49240, 1:1000), phosphorylated FUNDC1 (p-FUNDC1) (Affinity Biosciences, AF0001, 1:1000), total JNK (t-JNK) (Affinity Biosciences, AF6318, 1:1000), p-JNK (Affinity Biosciences, AF3318, 1:1000), IL-6 (Affinity Biosciences, DF6087, 1:1000), IL-8 (Wuhan Sanying Biotechnology Co, Ltd, 27095-1-AP, 1:1000), TNFα (Wuhan Sanying Biotechnology Co, Ltd, 26405-1-AP, 1:1000), MMP9 (Wuhan Sanying Biotechnology Co, Ltd, 27306-1-AP, 1:500), LC3 (Wuhan Sanying Biotechnology Co, Ltd, 14600-1-AP, 1:2500), Tomm20 (Wuhan Sanying Biotechnology Co, Ltd, 11802-1-AP, 1:8000), p62 (Affinity Biosciences, AF5384, 1:1000), cyt C (Wuhan Sanying Biotechnology Co, Ltd, 66264-1-Ig, 1:20000), and caspase-3 (Wuhan Sanying Biotechnology Co, Ltd, 19677-1-AP, 1:500).

Techniques: Activity Assay