N6-methyladenosine reader IGF2BP2 regulates NIPSNAP1-mediated mitophagy and mitochondrial dynamics to alleviate hepatic ischemia-reperfusion injury

Figure 1 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 expression in hepatic ischemia-reperfusion. ^aP < 0.05, ^bP < 0.01. A: Liver 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) protein levels at different time points following reperfusion, n = 3; B: NIPSNAP1 mRNA levels, n = 6; C: NIPSNAP1 protein expression in HepG2 cells exposed to hypoxia for 1 hour and after subsequent reoxygenation for 0 hour, 2 hours, 4 hours, 6 hours and 8 hours; n = 3; D: NIPSNAP1 mRNA levels in HepG2 cells subjected to hypoxia/reoxygenation; n = 6; E: Immunofluorescence staining of NIPSNAP1 localized in hepatocytes in the liver sections; scale bars, 20 μm (n = 6). NIPSNAP1: 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1; H/R: Hypoxia/reoxygenation.

Figure 2 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 regulates mitophagy in hepatic ischemia-reperfusion. ^aP < 0.05, ^bP < 0.01. A: Hematoxylin eosin staining, n = 6. Scale bar: 100 μm; B and C: Alanine aminotransferase and aspartate aminotransferase activities, n = 6; D: Mice were injected with adeno-associated virus-shRNA-4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and then subjected to hepatic ischemia-reperfusion. NIPSNAP1 protein, n = 3; E: Liver MAP1LC3 II/I and p62 proteins, n = 3; F and G: HepG2 cells were subjected to hypoxia/reoxygenation after transfection with NIPSNAP1 small interfering RNAs or pcDNA-NIPSNAP1. NIPSNAP1, p62 and MAP1LC3 II/I proteins, n = 3; H: Jung’s cyanine dye 1 staining. Scale bar, 50 μm. HE: Hematoxylin eosin; NIPSNAP1: 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1; AAV: Adeno-associated virus; NC: Negative control; LC3: MAP1LC3; I/R: Ischemia-reperfusion; H/R: Hypoxia/reoxygenation.

Figure 3 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 regulates mitochondrial dynamics in hepatic ischemia-reperfusion. ^aP < 0.05, ^bP < 0.01. A: Mitochondrial morphology was assessed using transmission electron microscopy (× 40000, magnification); B: Co-immunoprecipitation was used to verify the interaction between 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) and MFN2 or FIS1 in the livers of mice; C: Liver MFN2, FIS1, dynamin-related protein 1, and phosphorylated dynamin-related protein 1 (s616) proteins, n = 3; D and E: HepG2 cells were exposed to hypoxia/reoxygenation after transfection with NIPSNAP1 small interfering RNAs or pcDNA-NIPSNAP1. MFN2, FIS1, Drp1 and phosphorylated dynamin-related protein 1(s616) proteins, n = 3. AAV: Adeno-associated virus; I/R: Ischemia-reperfusion; NIPSNAP1: 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1; TEM: Transmission electron microscopy; NC: Negative control; IP: Immunoprecipitation; MFN2: Mitofusin 2; FIS1: Mitochondrial fission 1 protein; Drp1: Dynamin-related protein 1; p-Drp1: Phosphorylated dynamin-related protein 1; H/R: Hypoxia/reoxygenation.

Figure 4 Insulin-like growth factor 2 mRNA-binding protein 2 regulates 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 mRNA stability through n6-methyladenosine modification. ^aP < 0.05, ^bP < 0.01. A: The n6-methyladenosine enrichment of 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) mRNA in HepG2 cells was assessed by MeRIP^TM-quantitative real-time polymerase chain reaction, n = 6; B: Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) was pulled down and enriched with biotinylated NIPSNAP1 mRNA in HepG2 cells; C: IGF2BP2 RNA immunoprecipitation was used to assess the interaction between IGF2BP2 and NIPSNAP1 mRNA in HepG2 cells via quantitative real-time polymerase chain reaction, n = 6; D and E: HepG2 cells were subjected to hypoxia/reoxygenation after transfection with IGF2BP2 small interfering RNAs. NIPSNAP1 mRNA levels, n = 6. IGF2BP2 and NIPSNAP1 proteins, n = 3; F and G: HepG2 cells were subjected to hypoxia/reoxygenation after transfection with pcDNA-IGF2BP2. NIPSNAP1 mRNA levels, n = 6. IGF2BP2 and NIPSNAP1 proteins, n = 3; H: After HepG2 cells were transfected with si-control or si-IGF2BP2, they were treated with 2 μg/mL actinomycin D, and total RNA was extracted at different time intervals. NIPSNAP1: 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1; IGF2BP2: Insulin-like growth factor 2 mRNA-binding protein 2; IB: Immunoblotting; NC: Negative control; H/R: Hypoxia/reoxygenation.

Figure 5 The protective effect of interacted with insulin-like growing factor 2 overexpression in hepatic ischemia-reperfusion is suppressed by 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 knockdown. ^aP < 0.05, ^bP < 0.01. A: Hematoxylin eosin staining, n = 6. Scale bar: 100 μm; B: Alanine aminotransferase and aspartate aminotransferase activities, n = 6; C: Mitochondrial morphology was assessed using transmission electron microscopy (× 40000, magnification); D: Adeno-associated virus 9-mediated delivery was used to generate 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) shRNA - and interacted with insulin-like growing factor 2-overexpressing mice, which were subsequently subjected to ischemia-reperfusion. NIPSNAP1, MAP1LC3 II/I, p62, mitofusin 2, mitochondrial fission 1 protein, and phosphorylated dynamin-related protein 1/dynamin-related protein 1 (Drp1) proteins, n = 3; E: HepG2 cells were subjected to hypoxia/reoxygenation after cotransfection with pcDNA-interacted with insulin-like growing factor 2 and NIPSNAP1 small interfering RNAs. NIPSNAP1, MAP1LC3 II/I, p62, mitofusin 2, mitochondrial fission 1 protein, and phosphorylated-Drp1/Drp1 proteins, n = 3. HE: Hematoxylin eosin; NIPSNAP1: 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1; AAV: Adeno-associated virus; I/R: Ischemia-reperfusion; IGF2BP2: Insulin-like growth factor 2 mRNA-binding protein 2; TBG: Thyroxine-binding globulin; TEM: Transmission electron microscopy; MFN2: Mitofusin 2; FIS1: Mitochondrial fission 1 protein; Drp1: Dynamin-related protein 1; p-Drp1: Phosphorylated dynamin-related protein 1; LC3: MAP1LC3; NC: Negative control.