Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) knockout antagonised the protective effect of milk fat globule—epidermal growth factor 8 (MFG‐E8) on the in vitro acute pancreatitis (AP). (A) MFG‐E8 directly binds to ABCE1 (BioGRID database). (B) MFG‐E8 directly binds to ABCE1 in AR42J cells. (C) ATP levels in AR42J cells. (D) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (E) Representative images of FerroOrange (200×) in AR42J cells. (F) Representative images of Mito‐SOX and Mito‐Tracker red (1500×) in AR42J cells. (G and H) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (I and J) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (K) Representative images of BODIPY 581/591 (1500×) in AR42J cells. (L and M) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. (N) Lactate dehydrogenase (LDH) levels in supernatant. (O) Amylase levels in supernatant. n = 6, error bars indicate the SEM; * p < .05 versus vehicle‐AP (Lv‐vector); # p < .05 versus Lv‐ mfge8 ‐AP. Cer, cerulein; IB, immunoblotting; IP, immunoprecipitation; KO, knockout; LPS, lipopolysaccharide; Lv, lentivirus.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: Binding Assay, Knock-Out, In Vitro, Fluorescence, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Promoting the recovery of mitophagy alleviates the aggravation of acute pancreatitis (AP) in vitro caused by the milk fat globule—epidermal growth factor 8 (MFG‐E8)/adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) axis disorder. (A) Amylase levels in supernatant. (B) Lactate dehydrogenase (LDH) levels in supernatant. (C and D) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (E) Representative images of Mito‐SOX and Mito‐Tracker red (1500×) in AR42J cells. (F and G) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (H) ATP levels in AR42J cells. (I) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (J) Representative images of FerroOrange and BODIPY 581/591 (1500×) in AR42J cells. (K and L) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. n = 3‒6, error bars indicate the SEM; * p < .05 versus vehicle (Lv‐ Mfge8 + Abce1 ‐KO); # p < .05 versus vehicle‐AP. Cer, cerulein; FRAP, ferric reducing antioxidant power; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; Lv, lentivirus; MA‐5, mitochonic acid 5; KO, knockout.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: In Vitro, Binding Assay, Western Blot, Expressing, Fluorescence, Knock-Out

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) knockout antagonised the protective effect of milk fat globule—epidermal growth factor 8 (MFG‐E8) on the in vitro acute pancreatitis (AP). (A) MFG‐E8 directly binds to ABCE1 (BioGRID database). (B) MFG‐E8 directly binds to ABCE1 in AR42J cells. (C) ATP levels in AR42J cells. (D) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (E) Representative images of FerroOrange (200×) in AR42J cells. (F) Representative images of Mito‐SOX and Mito‐Tracker red (1500×) in AR42J cells. (G and H) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (I and J) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (K) Representative images of BODIPY 581/591 (1500×) in AR42J cells. (L and M) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. (N) Lactate dehydrogenase (LDH) levels in supernatant. (O) Amylase levels in supernatant. n = 6, error bars indicate the SEM; * p < .05 versus vehicle‐AP (Lv‐vector); # p < .05 versus Lv‐ mfge8 ‐AP. Cer, cerulein; IB, immunoblotting; IP, immunoprecipitation; KO, knockout; LPS, lipopolysaccharide; Lv, lentivirus.

Article Snippet: The mfge8 gene knockout ( mfge8 ‐KO) mice were created through the CRISPR/Cas9 method (Shanghai Model Organisms Center, Inc.), which involved the deletion of exons 2‒6 of the mfge8 gene on a C57BL/6J genetic background.

Techniques: Binding Assay, Knock-Out, In Vitro, Fluorescence, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Promoting the recovery of mitophagy alleviates the aggravation of acute pancreatitis (AP) in vitro caused by the milk fat globule—epidermal growth factor 8 (MFG‐E8)/adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) axis disorder. (A) Amylase levels in supernatant. (B) Lactate dehydrogenase (LDH) levels in supernatant. (C and D) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (E) Representative images of Mito‐SOX and Mito‐Tracker red (1500×) in AR42J cells. (F and G) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (H) ATP levels in AR42J cells. (I) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (J) Representative images of FerroOrange and BODIPY 581/591 (1500×) in AR42J cells. (K and L) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. n = 3‒6, error bars indicate the SEM; * p < .05 versus vehicle (Lv‐ Mfge8 + Abce1 ‐KO); # p < .05 versus vehicle‐AP. Cer, cerulein; FRAP, ferric reducing antioxidant power; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; Lv, lentivirus; MA‐5, mitochonic acid 5; KO, knockout.

Article Snippet: The mfge8 gene knockout ( mfge8 ‐KO) mice were created through the CRISPR/Cas9 method (Shanghai Model Organisms Center, Inc.), which involved the deletion of exons 2‒6 of the mfge8 gene on a C57BL/6J genetic background.

Techniques: In Vitro, Binding Assay, Western Blot, Expressing, Fluorescence, Knock-Out

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Overexpression of milk fat globule—epidermal growth factor 8 (MFG‐E8) restores mitophagy in experimental acute pancreatitis (AP). (A) Western blot analysis of the MFG‐E8 expression level in AR42J cells. (B) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (C) Representative images of Mito‐SOX (1500×) and Mito‐Tracker red (1500×) in AR42J cells. (D and E) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (F) Adenosine triphosphate (ATP) levels in AR42J cells. (G) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (H) Representative images of JC‐1 (1500×) in AR42J cells. (I) Western blot analysis of the peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α), mitochondrial transcription factor A (Tfam), fission protein 1 (Fis‐1) and dynamin‐related protein 1 (Drp‐1) expression level in AR42J cells. n = 6, error bars indicate the SEM; * p < .05 versus Lv‐vector‐PBS; # p < .05 versus Lv‐vector‐AP. Cer, cerulein; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; Lv, lentivirus.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: Over Expression, Western Blot, Expressing, Fluorescence, Plasmid Preparation

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Overexpression of milk fat globule—epidermal growth factor 8 (MFG‐E8) alleviates ferroptosis in experimental acute pancreatitis (AP). (A) Malondialdehyde (MDA) levels in AR42J cells. (B) Glutathione (GSH) levels in AR42J cells. (C) Representative images of FerroOrange (1500×) in AR42J cells. (D) Representative images of BODIPY 581/591 (1500×) in AR42J cells. (E and F) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. (G) Lactate dehydrogenase (LDH) levels in supernatant. (H) Amylase levels in supernatant. n = 6, error bars indicate the SEM; * p < .05 versus Lv‐vector‐PBS; # p < .05 versus Lv‐vector‐AP. Cer, cerulein; LPS, lipopolysaccharide; Lv, lentivirus.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: Over Expression, Western Blot, Expressing, Plasmid Preparation

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) knockout antagonised the protective effect of milk fat globule—epidermal growth factor 8 (MFG‐E8) on the in vitro acute pancreatitis (AP). (A) MFG‐E8 directly binds to ABCE1 (BioGRID database). (B) MFG‐E8 directly binds to ABCE1 in AR42J cells. (C) ATP levels in AR42J cells. (D) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (E) Representative images of FerroOrange (200×) in AR42J cells. (F) Representative images of Mito‐SOX and Mito‐Tracker red (1500×) in AR42J cells. (G and H) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (I and J) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (K) Representative images of BODIPY 581/591 (1500×) in AR42J cells. (L and M) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. (N) Lactate dehydrogenase (LDH) levels in supernatant. (O) Amylase levels in supernatant. n = 6, error bars indicate the SEM; * p < .05 versus vehicle‐AP (Lv‐vector); # p < .05 versus Lv‐ mfge8 ‐AP. Cer, cerulein; IB, immunoblotting; IP, immunoprecipitation; KO, knockout; LPS, lipopolysaccharide; Lv, lentivirus.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: Binding Assay, Knock-Out, In Vitro, Fluorescence, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Promoting the recovery of mitophagy alleviates the aggravation of acute pancreatitis (AP) in vitro caused by the milk fat globule—epidermal growth factor 8 (MFG‐E8)/adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) axis disorder. (A) Amylase levels in supernatant. (B) Lactate dehydrogenase (LDH) levels in supernatant. (C and D) Western blot analysis of the PINK1, Mitofusin 2 (MFN2) and Parkin expression level in AR42J cells. (E) Representative images of Mito‐SOX and Mito‐Tracker red (1500×) in AR42J cells. (F and G) Relative fluorescence intensity of Mito‐SOX and Mito‐Tracker red in AR42J cells. (H) ATP levels in AR42J cells. (I) Ferric reducing antioxidant power (FRAP) levels in AR42J cells. (J) Representative images of FerroOrange and BODIPY 581/591 (1500×) in AR42J cells. (K and L) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. n = 3‒6, error bars indicate the SEM; * p < .05 versus vehicle (Lv‐ Mfge8 + Abce1 ‐KO); # p < .05 versus vehicle‐AP. Cer, cerulein; FRAP, ferric reducing antioxidant power; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; Lv, lentivirus; MA‐5, mitochonic acid 5; KO, knockout.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: In Vitro, Binding Assay, Western Blot, Expressing, Fluorescence, Knock-Out

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Inhibiting ferroptosis alleviates the aggravation of acute pancreatitis (AP) in vitro caused by the milk fat globule—epidermal growth factor 8 (MFG‐E8)/adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1) axis disorder. (A) Supernatant lactate dehydrogenase (LDH) levels. (B) Supernatant amylase levels. (C and D) Western blot analysis of the solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1) and glutathione peroxidase (GPX4) expression level in AR42J cells. (E) Representative images of BODIPY 581/591 (1500×) in AR42J cells. (F) Supernatant LDH levels. (G) Supernatant amylase levels. (H and I) Western blot analysis of the SLC7A11, FTH1 and GPX4 expression level in AR42J cells. (J) Representative images of BODIPY 581/591 (1500×) in AR42J cells. n = 3‒6, error bars indicate the SEM; * p < .05 versus 0 µM Ferrostatin‐1 (Fer‐1) or 0 nM Liproxstatin‐1 (Lip‐1); # p < .05 versus 5 µM Fer‐1 or 40 nM Lip‐1. Cer, cerulein; KO, knockout; LPS, lipopolysaccharide; Lv, lentivirus.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: In Vitro, Binding Assay, Western Blot, Expressing, Knock-Out

Journal: Clinical and Translational Medicine

Article Title: Milk fat globule—EGF factor 8/ATP‐binding cassette subfamily E member 1 axis maintains mitophagy flux homeostasis to suppress ferroptosis in acute pancreatitis

doi: 10.1002/ctm2.70619

Figure Lengend Snippet: Graphical abstract. During acute pancreatitis (AP), milk fat globule—epidermal growth factor 8 (MFG‐E8) expression in pancreatic acinar cells is downregulated, resulting in disruption of the adenosine triphosphate (ATP)‐binding cassette subfamily E member 1 (ABCE1)/MFG‐E8 complex. This impairment leads to defective mitophagy flux, accumulation of lipid peroxides and ferroptosis, ultimately contributing to pancreatic damage.

Article Snippet: On the basis of this cell line, we further transfected it with MFG‐E8‐overexpressing lentivirus to achieve the overexpression of mfge8 gene in abce1 ‐KO AR42J monoclonal cells (Lv‐ mfge8 + abce1 ‐KO, dual genetic modification, Soochow Cyagen Corporation).

Techniques: Expressing, Disruption, Binding Assay