Journal: International Journal of Medical Sciences

Article Title: B355252, A Novel Small Molecule, Confers Neuroprotection Against Cobalt Chloride Toxicity In Mouse Hippocampal Cells Through Altering Mitochondrial Dynamics And Limiting Autophagy Induction

doi: 10.7150/ijms.24702

Figure Lengend Snippet: B355252 protects against CoCl 2 -induced alterations in mitochondrial fusion proteins OPA1 and Mfn2 A) Western blot of mitochondrial fusion protein, OPA1, and loading control, β-actin, in HT22 cells. Total protein lysates were obtained from untreated control and cells treated for 10 and 24hr with 300 μM CoCl 2 with or without 2.5 μM B355252. Picture is representative of three separate experiments. B) Quantification of average OPA1 expression when normalized to β-actin. C) Western blot of mitochondrial fusion protein, Mfn2, and loading control, β-actin, in HT22 cells. Protein samples were obtained as indicated in A and picture is representative of three separate experiments. D) Quantification of average Mfn2 expression when normalized to β-actin. One-way ANOVA followed by Bonferroni's post-test was used for analysis. ### p<0.001 vs. control. *** p<0.001 vs. CoCl 2 alone.

Article Snippet: Purified Mouse Anti-OPA1 monoclonal antibody was obtained from BD Transduction Laboratories (Franklin Lake, NJ).

Techniques: Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: ALCAT1-mediated abnormal cardiolipin remodelling promotes mitochondrial injury in podocytes in diabetic kidney disease

doi: 10.1186/s12964-023-01399-4

Figure Lengend Snippet: ALCAT1 inhibition or SS-31 treatment attenuated abnormal cardiolipin remodelling and podocyte mitochondrial damage in diabetic mice. A Relative content of oxidized cardiolipin in the renal cortex ( n = 6, ** p <0.01, *** p <0.001). B Micrographs and semiquantitative DHE staining in different groups ( n = 6, * p <0.05, ** p <0.01, scale bars: 30 μm). C Representative electron microscopy images of the ultrastructure of podocyte mitochondria from different groups ( n = 6, * p <0.05, ** p <0.01, scale bars: 1 μm). D Western blotting in the glomerulus of ALCAT1, mitochondrial fusion/fission-related proteins (FIS1, DRP1, OPA1, MFN2), autophagy-related proteins (PINK1, LC3B, P62), and apoptosis-related proteins (BCL2, BAX, cleaved caspase-3) ( n = 6, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, ns p >0.05)

Article Snippet: Western blotting was performed using the following antibodies: anti-ALCAT1 (1:1000, Invitrogen, USA), anti-β-actin (1:2000, Proteintech, China), anti-fission 1 (1:1000, FIS1; GeneTex, USA), anti-dynamin-related protein-1 (1:1000, DRP1; ImmunoWay, USA), anti-optic atrophy-1 (1:1000, OPA1; ImmunoWay, USA), anti-mitofusin2 (1:1000, MFN2; GeneTex, USA), anti-B-cell lymphoma-2 (1:1000, BCL2; Cell Signaling Technology, USA), anti-BCL2-Associated X (1:1000, BAX; Cell Signaling Technology, USA), anti-Caspase3 (1:1000, Cell Signaling Technology, USA), anti-PINK1 (1:1000, Novus, USA), anti-LC3B (1:1000, Abcam, USA), anti-P62 (1:1000, GeneTex, USA), anti-AMPK (1:1000, ImmunoWay, USA), anti-phospho-AMPK (1:1000, ImmunoWay, USA), and HRP-linked goat anti-rabbit/mouse IgG (1:10000, Antgene, China).

Techniques: Inhibition, Staining, Electron Microscopy, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: ALCAT1-mediated abnormal cardiolipin remodelling promotes mitochondrial injury in podocytes in diabetic kidney disease

doi: 10.1186/s12964-023-01399-4

Figure Lengend Snippet: ALCAT1 was increased in HG-treated podocytes, accompanied by abnormal cardiolipin remodelling and mitochondrial dysfunction. A Western blotting was used to measure the variations in ALCAT1 levels in cultured podocytes subjected to glucose stimulation with time gradients (0, 6, 12, 24, 36, and 48 h) ( n = 3, ** p <0.01, **** p <0.0001). B Representative immunofluorescence staining for ALCAT1 (red) and semiquantitative analysis of these results in each group ( n = 3, *** p <0.001, scale bars: 40 μm). C Relative content of oxidized cardiolipin ( n = 3, *** p <0.001). D Cellular ROS and mitochondrial ROS production were assessed by DCFH-DA fluorescent probe and MitoSox Red fluorescence staining in different groups ( n = 3, ** p <0.01, *** p <0.001, scale bars: 200 μm). E Determination of relative ATP content in each group ( n = 3, ** p <0.01). F Relative quantification of mitochondrial membrane potential by JC-1 staining in podocytes ( n = 3, *** p <0.001, scale bars: 200 μm). G Microscopy images of mitochondrial fission and fusion by MitoTracker Red staining and semiquantitative analysis of the average mitochondrial length and aspect ratio ( n = 3, ** p <0.01, *** p <0.001, scale bars: 40 μm). H TEM results of mitochondrial structure damage in podocytes ( n = 3, * p <0.05, scale bars: 1 μm). I Apoptosis detection by flow cytometry in podocytes ( n = 3, **** p <0.0001). J Western blotting of mitochondrial fusion/fission-related proteins (FIS1, DRP1, OPA1, MFN2), autophagy-related proteins (PINK1, LC3B, P62), and apoptosis-related proteins (BCL2, BAX, cleaved caspase-3) ( n = 3, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001)

Article Snippet: Western blotting was performed using the following antibodies: anti-ALCAT1 (1:1000, Invitrogen, USA), anti-β-actin (1:2000, Proteintech, China), anti-fission 1 (1:1000, FIS1; GeneTex, USA), anti-dynamin-related protein-1 (1:1000, DRP1; ImmunoWay, USA), anti-optic atrophy-1 (1:1000, OPA1; ImmunoWay, USA), anti-mitofusin2 (1:1000, MFN2; GeneTex, USA), anti-B-cell lymphoma-2 (1:1000, BCL2; Cell Signaling Technology, USA), anti-BCL2-Associated X (1:1000, BAX; Cell Signaling Technology, USA), anti-Caspase3 (1:1000, Cell Signaling Technology, USA), anti-PINK1 (1:1000, Novus, USA), anti-LC3B (1:1000, Abcam, USA), anti-P62 (1:1000, GeneTex, USA), anti-AMPK (1:1000, ImmunoWay, USA), anti-phospho-AMPK (1:1000, ImmunoWay, USA), and HRP-linked goat anti-rabbit/mouse IgG (1:10000, Antgene, China).

Techniques: Western Blot, Cell Culture, Immunofluorescence, Staining, Fluorescence, Membrane, Microscopy, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: ALCAT1-mediated abnormal cardiolipin remodelling promotes mitochondrial injury in podocytes in diabetic kidney disease

doi: 10.1186/s12964-023-01399-4

Figure Lengend Snippet: ALCAT1 deficiency improved abnormal cardiolipin remodelling and suppressed mitochondrial damage in high glucose-treated podocytes. A Verification of the knockdown effect of ALCAT1 siRNA by Western blotting ( n = 3, *** p <0.001). B Immunofluorescence staining of ALCAT1 (red) in podocytes from each group ( n = 3, *** p <0.001, scale bars: 40 μm). C Relative content of oxidized cardiolipin in podocytes measured by lipidomic analysis ( n = 3, *** p <0.001). D Cellular ROS and mitochondrial ROS production were evaluated by DCFH-DA fluorescent probe and MitoSox Red fluorescence staining ( n = 3, * p <0.05, **p<0.01, scale bars: 200 μm). E Relative ATP content and semiquantitative analysis of each group ( n = 3, ** p <0.01). F Relative quantification of mitochondrial membrane potential detected by JC-1 staining ( n = 3, ** p <0.01, scale bars: 200 μm). G Microscopy images of mitochondrial fission and fusion in podocytes and semiquantitative analysis of the average mitochondrial length and aspect ratio ( n = 3, ** p <0.01, *** p <0.001, scale bars: 60 μm). H Electron microscopy observation of the ultrastructure of mitochondria in podocytes ( n = 3, ** p <0.01, scale bars: 2 μm). I Apoptosis detection in each group by flow cytometry ( n = 3, *** p <0.001). J Western blot analysis of mitochondrial fusion/fission-related proteins (FIS1, DRP1, OPA1, MFN2), autophagy-related proteins (PINK1, LC3B, P62), and apoptosis-related proteins (BCL2, BAX, cleaved caspase-3) ( n = 3, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001)

Article Snippet: Western blotting was performed using the following antibodies: anti-ALCAT1 (1:1000, Invitrogen, USA), anti-β-actin (1:2000, Proteintech, China), anti-fission 1 (1:1000, FIS1; GeneTex, USA), anti-dynamin-related protein-1 (1:1000, DRP1; ImmunoWay, USA), anti-optic atrophy-1 (1:1000, OPA1; ImmunoWay, USA), anti-mitofusin2 (1:1000, MFN2; GeneTex, USA), anti-B-cell lymphoma-2 (1:1000, BCL2; Cell Signaling Technology, USA), anti-BCL2-Associated X (1:1000, BAX; Cell Signaling Technology, USA), anti-Caspase3 (1:1000, Cell Signaling Technology, USA), anti-PINK1 (1:1000, Novus, USA), anti-LC3B (1:1000, Abcam, USA), anti-P62 (1:1000, GeneTex, USA), anti-AMPK (1:1000, ImmunoWay, USA), anti-phospho-AMPK (1:1000, ImmunoWay, USA), and HRP-linked goat anti-rabbit/mouse IgG (1:10000, Antgene, China).

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence, Membrane, Microscopy, Electron Microscopy, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: ALCAT1-mediated abnormal cardiolipin remodelling promotes mitochondrial injury in podocytes in diabetic kidney disease

doi: 10.1186/s12964-023-01399-4

Figure Lengend Snippet: Inhibition of cardiolipin oxidation by SS-31 reversed ALCAT1 upregulation-mediated abnormal cardiolipin remodelling and mitochondrial damage in HG-induced podocytes. A The effects of overexpression plasmid transfection and SS-31 treatment on ALCAT1 expression were detected by Western blotting ( n = 3, **** p <0.0001, ns p >0.05). B Immunofluorescence staining of ALCAT1 (red) in podocytes from each group ( n = 3, *** p <0.001, ns p >0.05, scale bars: 20 μm). C Relative oxidized cardiolipin content in podocytes measured by lipidomic analysis ( n = 3, ** p <0.01, *** p <0.001). D Cellular ROS and mitochondrial ROS production were evaluated by DCFH-DA fluorescent probe and MitoSox Red fluorescence staining ( n = 3, * p <0.05, ** p <0.01, **** p <0.0001, scale bars: 200 μm). E Relative ATP content and semiquantitative analysis of each group ( n = 3, * p <0.05). F Relative quantification of mitochondrial membrane potential detected by JC-1 staining ( n = 3, *** p <0.001, scale bars: 200 μm). G Microscopy images of mitochondrial fission and fusion in podocytes by MitoTracker Red staining and semiquantitative analysis of the average mitochondrial length and aspect ratio ( n = 3, * p <0.05, ** p <0.01, scale bars: 40 μm). H Electron microscopy observation of the ultrastructure of mitochondria in podocytes ( n = 3, * p <0.05, *** p <0.001, scale bars: 2 μm). I Apoptosis detection in each group by flow cytometry ( n = 3, ** p <0.01, *** p <0.001). J Western blot analysis of mitochondrial fusion/fission-related proteins (FIS1, DRP1, OPA1, MFN2), autophagy-related proteins (PINK1, LC3B, P62), and apoptosis-related proteins (BCL2, BAX, cleaved caspase-3) ( n = 3, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001, ns p >0.05)

Article Snippet: Western blotting was performed using the following antibodies: anti-ALCAT1 (1:1000, Invitrogen, USA), anti-β-actin (1:2000, Proteintech, China), anti-fission 1 (1:1000, FIS1; GeneTex, USA), anti-dynamin-related protein-1 (1:1000, DRP1; ImmunoWay, USA), anti-optic atrophy-1 (1:1000, OPA1; ImmunoWay, USA), anti-mitofusin2 (1:1000, MFN2; GeneTex, USA), anti-B-cell lymphoma-2 (1:1000, BCL2; Cell Signaling Technology, USA), anti-BCL2-Associated X (1:1000, BAX; Cell Signaling Technology, USA), anti-Caspase3 (1:1000, Cell Signaling Technology, USA), anti-PINK1 (1:1000, Novus, USA), anti-LC3B (1:1000, Abcam, USA), anti-P62 (1:1000, GeneTex, USA), anti-AMPK (1:1000, ImmunoWay, USA), anti-phospho-AMPK (1:1000, ImmunoWay, USA), and HRP-linked goat anti-rabbit/mouse IgG (1:10000, Antgene, China).

Techniques: Inhibition, Over Expression, Plasmid Preparation, Transfection, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence, Membrane, Microscopy, Electron Microscopy, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: ALCAT1-mediated abnormal cardiolipin remodelling promotes mitochondrial injury in podocytes in diabetic kidney disease

doi: 10.1186/s12964-023-01399-4

Figure Lengend Snippet: Aberrant cardiolipin remodelling mediated by ALCAT1 promoted mitochondrial malfunction in podocytes by deactivating the AMPK pathway. A Western blotting analysis of ALCAT1, AMPK and p-AMPK in vitro, as well as semi-quantitative of pAMPK/AMPK ratios ( n = 3, *** p <0.001, ns p >0.05). B Western blotting analysis of ALCAT1, AMPK and p-AMPK in vivo, as well as semi-quantitative of pAMPK/AMPK ratios ( n = 3, ** p <0.01, ns p >0.05). C Western blot analysis of mitochondrial fusion/fission-related proteins (FIS1, DRP1, OPA1, MFN2), autophagy-related proteins (PINK1, LC3B, P62), and apoptosis-related proteins (BCL2, BAX, cleaved caspase-3) in vitro ( n = 3, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001). D Western blot analysis of mitochondrial fusion/fission-related proteins (FIS1, DRP1, OPA1, MFN2), autophagy-related proteins (PINK1, LC3B, P62), and apoptosis-related proteins (BCL2, BAX, cleaved caspase-3) in vivo ( n = 3, * p <0.05, ** p <0.01, *** p <0.001). E Schematic of the molecular model proposed in this study

Article Snippet: Western blotting was performed using the following antibodies: anti-ALCAT1 (1:1000, Invitrogen, USA), anti-β-actin (1:2000, Proteintech, China), anti-fission 1 (1:1000, FIS1; GeneTex, USA), anti-dynamin-related protein-1 (1:1000, DRP1; ImmunoWay, USA), anti-optic atrophy-1 (1:1000, OPA1; ImmunoWay, USA), anti-mitofusin2 (1:1000, MFN2; GeneTex, USA), anti-B-cell lymphoma-2 (1:1000, BCL2; Cell Signaling Technology, USA), anti-BCL2-Associated X (1:1000, BAX; Cell Signaling Technology, USA), anti-Caspase3 (1:1000, Cell Signaling Technology, USA), anti-PINK1 (1:1000, Novus, USA), anti-LC3B (1:1000, Abcam, USA), anti-P62 (1:1000, GeneTex, USA), anti-AMPK (1:1000, ImmunoWay, USA), anti-phospho-AMPK (1:1000, ImmunoWay, USA), and HRP-linked goat anti-rabbit/mouse IgG (1:10000, Antgene, China).

Techniques: Western Blot, In Vitro, In Vivo