Journal: Cell Death & Disease

Article Title: Macrophage migration inhibitory factor (MIF) suppresses mitophagy through disturbing the protein interaction of PINK1-Parkin in sepsis-associated acute kidney injury

doi: 10.1038/s41419-024-06826-z

Figure Lengend Snippet: The qPCR was utilized to assess the relative expression levels of ( a ) PRKN mRNA and ( b ) PINK1 mRNA, comparing the si- Nctrl group with the si- PRKN or si- PINK1 group. Additionally, c the levels of MIF, Casp3, cleaved-Casp3, and GAPDH in the cytoplasmic fraction (Cyto), as well as PINK1, Parkin, and VDAC1 in the mitochondrial enriched fraction (Mito), were determined via immunoblotting. The relative expression ratios of these proteins were quantified: d cleaved-Casp3/GAPDH, e MIF/GAPDH, f PINK1/VDAC1, and g Parkin/VDAC1. h Representative images from triple-labeling immunofluorescence of various HK-2 cell groups were analyzed. The potential mitophagosomes, based on the colocalization of LC3 (green), VDAC1 (red), and LAMP1 (purple), were identified by white arrows, while areas without colocalization were presented within yellow boxes. Scale bar: 10 μm. i The presence of mitophagosome formation in HK-2 cells was assessed using triple-labeling immunofluorescence images. j Representative TEM images depicted the morphology of mitophagosomes (mitochondria-like structures enclosed by autophagosomes, marked by red arrows). Scale bar: 500 nm. k Apoptosis in HK-2 cells, stained with annexin V-FITC and PI, was quantified using FCM. l The mean number of mitophagosomes per 25 μm 2 was calculated from ten random fields of TEM view. m The percentage of apoptotic cells was determined. Data were presented as mean ± SEM. n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: A batch of cells or a segment of renal tissues was incubated with a mitochondria isolation reagent (MedChemExpress, HY-K1060, and HY-K1061) on ice and subsequently homogenized.

Techniques: Expressing, Western Blot, Labeling, Immunofluorescence, Staining

Journal: Cell Death & Disease

Article Title: Macrophage migration inhibitory factor (MIF) suppresses mitophagy through disturbing the protein interaction of PINK1-Parkin in sepsis-associated acute kidney injury

doi: 10.1038/s41419-024-06826-z

Figure Lengend Snippet: a , b Hematoxylin and eosin (H&E) staining was performed on renal tissues, and representative images were obtained to evaluate the percentage of tubule damage in each group. Scale bar: 50 μm. ( c , d ) Immunohistochemical staining using MIF antibody was conducted on renal tissues, and representative images were captured to calculate the percentage of the area stained with MIF in different groups. Scale bar: 50 μm. e , f Immunohistochemical staining with cleaved-casp3 antibody was performed on renal tissues, and representative images were acquired to determine the percentage of the area stained with cleaved-casp3 in different groups. Scale bar: 50 μm. g , h Renal tissues were subjected to immunofluorescence staining with TUNEL, and representative images were taken to quantify the number of TUNEL-positive cells per 0.25 mm 2 in different groups. Scale bar: 50 μm. i Representative TEM images displayed the morphology of mitophagosomes (mitochondria-like structures enclosed by autophagosomes, indicated by red arrows). Scale bar: 500 nm. j The mean number of mitophagosomes per 25 μm 2 was calculated from ten random fields observed in TEM view. k Immunoblotting was utilized to measure the expression levels of MIF, Casp3, cleaved-Casp3, and β-Actin in the cytoplasmic fraction (Cyto), as well as PINK1, Parkin, and VDAC1 in the mitochondrial enriched fraction (Mito). The relative expression ratios of these proteins were quantified: l cleaved-Casp3/β-Actin, m MIF/β-Actin, n PINK1/VDAC1, and o Parkin/VDAC1. Data were presented as mean ± SEM. n = 6. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: A batch of cells or a segment of renal tissues was incubated with a mitochondria isolation reagent (MedChemExpress, HY-K1060, and HY-K1061) on ice and subsequently homogenized.

Techniques: Staining, Immunohistochemical staining, Immunofluorescence, TUNEL Assay, Western Blot, Expressing

Journal: Cell Death & Disease

Article Title: Macrophage migration inhibitory factor (MIF) suppresses mitophagy through disturbing the protein interaction of PINK1-Parkin in sepsis-associated acute kidney injury

doi: 10.1038/s41419-024-06826-z

Figure Lengend Snippet: (1) LPS triggered mitochondrial damage. (2) Typically, PINK1 relocated to the impaired mitochondria and recruited Parkin. (3) Parkin interacted with PINK1 and facilitated the formation of mitophagosomes, leading to the degradation of damaged mitochondria. T0467 enhanced the recruitment of Parkin to mitochondria. (4) However, LPS induced the upregulation of MIF. Elevated MIF interacted with PINK1 before Parkin, a process reversible by ISO-1. (5) This interaction hindered the formation of mitophagosomes, resulting in RTECs injury in SA-AKI. Generally, ISO-1 and T0467 both mitigated RTECs injury and improved renal function in SA-AKI by alleviating the inhibitory effect of MIF on the PINK1-Parkin pathway of mitophagy (By Figdraw).

Article Snippet: A batch of cells or a segment of renal tissues was incubated with a mitochondria isolation reagent (MedChemExpress, HY-K1060, and HY-K1061) on ice and subsequently homogenized.

Techniques: