Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 1 Imbalanced mitochondrial homeostasis is associated with renal proximal tubular injury in DKD. A DHE staining was performed to evaluate the production of ROS in the kidneys of the mice. B Representative images of SOD2 staining of kidneys from mice. C and D Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fractions of the mice (C) and HK-2 cells (D) as indicated. E and F H2DCFDA (E) and Mitosox (F) staining were performed to evaluate the generation of ROS in the cytoplasm and mitochondria of HK-2 cells, respectively. G Repre sentative images of MitoTracker Red-stained cells showing mitochondrial morphology in the indicated groups of cells. Scale bar = 100 μm in A and B for fluorescence images, 50 μm for images in E and F, and 20 μm for confocal images in G. For all statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Staining, Fluorescence

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 2 CaMKKβ-mediated protective effect on tubular injury in DKD. A IHC staining was performed to evaluate the protein expression of CaMKKβ in kid neys of the mice. Scale bar = 200 μm. B and C Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in the groups of mice (B) and HK-2 cells (C) cultured with HG as indicated. D IF staining of CaMKKβ in HK-2 cells cultured with HG for 48 h. Scale bar = 20 μm. E Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in HK-2 cells transfected with CaMKKβ as indicated. F IF staining was performed to evaluate the uptake of BSA-TRITC in HK-2 cells. Scale bar = 50 μm. G H2DCFDA and Mitosox staining were performed to evaluate the generation of ROS in the cytoplasm and mitochondria of HK-2 cells, respectively. Scale bar = 20 μm. H Representative images of MitoTracker Red-stained cells showing mitochondrial morphology in the indicated groups of cells. I Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fraction of HK-2 cells transfected with CaMKKβ as indicated. Scale bar = 20 μm. OE-NC represents the control of CaMKKβ. OE-CaMKKβ represents the overexpression of CaMKKβ. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Immunohistochemistry, Expressing, Cell Culture, Staining, Transfection, Control, Over Expression

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 5 Inhibition of NEDD4L ameliorates oxidative stress and mitochondrial dysfunction in diabetic mice. A Representative images of HE and PAS stain ing of kidney sections. B Representative images of Masson staining of kidney sections. C Representative images of DHE-stained kidney sections showing oxidative stress in the mice. A-C: Scale bar = 200 μm. D Representative immunoreactive bands for Bax, Bcl-2, and cleaved Caspase-3 in the groups of mice. E Representative immunoreactive bands for CaMKKβ, phosphorylated AMPK, and total AMPK in the groups of mice. F Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in different groups of mice, as indicated. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Inhibition, Staining

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 6 Silencing NEDD4L prevents HG-induced mitochondrial dysfunction in HK-2 cells. A IF staining was performed to evaluate the uptake of BSA-TRITC in the experiments. B H2DCFDA and Mitosox staining were performed to evaluate the generation of ROS in the cytoplasm and mitochondria of HK-2 cells, respectively. Scale bar = 50 μm. C Representative images of MitoTracker Red-stained cells showing mitochondrial morphology in the indicated groups of cells. Scale bar = 20 μm. D Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fraction of HK-2 cells transfected with Nedd4L-siRNA as indicated. E Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in HK-2 cells transfected with Nedd4L-siRNA as indicated. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Staining, Transfection

Journal: Journal of translational medicine

Article Title: Aberrant expression of NEDD4L disrupts mitochondrial homeostasis by downregulating CaMKKβ in diabetic kidney disease.

doi: 10.1186/s12967-024-05207-6

Figure Lengend Snippet: Fig. 7 Knockdown of NEDD4L contributes to HK-2 cell function via CaMKKβ/AMPK pathway. A Representative immunoreactive bands for CaMKKβ, AMPK, and phosphorylated AMPK in HK-2 cells cotransfected with Nedd4L-siRNA and CaMKKβ-siRNA as indicated. B Representative immunoreactive bands for Drp1, phosphorylated Drp1, Mfn2, and HSP60 in the mitochondrial fraction of HK-2 cells, as indicated. C MitoTracker Red staining showing mitochondrial morphology in the indicated groups of cells. Scale bar = 20 μm. D IF staining was performed to evaluate the uptake of BSA-TRITC in the indicated groups of cells. Scale bar = 50 μm. E and F H2DCFDA (E) and Mitosox (F) staining were performed to evaluate the generation of ROS in the cytoplasm and mito chondria of HK-2 cells, respectively. Scale bar = 50 μm. For all the statistical plots, the data are presented as the means ± SEMs. *P < 0.05

Article Snippet: The primary antibodies and their respective dilutions were NEDD4L, p-AMPK, AMPK, DRP-1, and HSP60 (all with a dilution ratio of 1:1000, Cell Signaling Technology, Danvers, USA), CaMKKβ and MFN-2 (1:1000 dilution, Abcam, Cambridge, UK), and phospho-DRP1 (Ser616) (1:1000 dilution, Biorbyt, Cambridge, UK).

Techniques: Knockdown, Cell Function Assay, Staining

Journal: Journal of Nanobiotechnology

Article Title: Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics

doi: 10.1186/s12951-024-02818-5

Figure Lengend Snippet: Impaired Drp1-mediated mitochondrial dynamism induces a senescent-like phenomenon in BMSCs. a Colocalization analysis of Drp1 (green) and mitochondria (red). Scale bar, 10 μm. MOC were analyzed by Zeiss LSM 980. b RT-qPCR assay of Drp1. c Representative images of SA-β-gal positive BMSCs. Scale bar, 50 μm. d Quantification of SA-β-gal positive BMSCs. e Representative images of EdU-positive BMSCs. Scale bar, 50 μm. f Quantification of EdU-positive BMSCs. g Representative images of ALP staining of BMSCs treated as indicated after osteogenic induction for 7 days. Data is shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: not significant ( p > 0.05)

Article Snippet: After fixation and blocking, cells were incubated with rabbit anti-human Drp1 primary antibody (1:100, BOSTER) in a wet box overnight at 4 °C.

Techniques: Quantitative RT-PCR, Staining

Journal: Journal of Nanobiotechnology

Article Title: Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics

doi: 10.1186/s12951-024-02818-5

Figure Lengend Snippet: Young sEVs rejuvenate replicative senescent BMSCs by promoting Drp1 translocation onto mitochondria. a SER texture analysis in BMSCs. Scale bar, 50 μm. b Analysis of mitochondrial membrane potential (MMP) using JC-1 staining. Quantification of depolarized cells was calculated. c Quantification of ATP levels per group. d Quantification of the intercellular ROS levels using mean fluorescence intensity of the DCFH-DA. e Quantification of the mitoSOX-positive mitochondria in all mitochondria. f Representative images of western blot analysis showing the expression levels of Drp1 and β-actin. g Representative images of colocalization of mitochondria (red), and Drp1 (green) per group. Scale bar, 10 μm. h Representative images of western blot analysis showing the change of mitochondrial fission gene Drp1. Data is shown as mean ± SEM. Comparison with P3 BMSCs: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns: no significant difference. Comparisons between identical passage: # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001. NS: no significant difference

Article Snippet: After fixation and blocking, cells were incubated with rabbit anti-human Drp1 primary antibody (1:100, BOSTER) in a wet box overnight at 4 °C.

Techniques: Translocation Assay, Membrane, Staining, Fluorescence, Western Blot, Expressing, Comparison