Journal: The Journal of Biological Chemistry

Article Title: Activation of mitophagy antagonizes high uric acid–induced hepatic lipid accumulation

doi: 10.1016/j.jbc.2025.111054

Figure Lengend Snippet: High uric acid (HUA) induces mitophagy in hepatocytes . A , representative Western blot (WB) images of PINK1, LC3B, and COXⅣ protein expression in primary mouse hepatocytes treated with HUA for different durations (0, 6, 12, 24, and 48 h). B – D , quantification of PINK1, LC3B-II/I, and COXⅣ protein levels in primary hepatocytes (n = 3). E , representative WB images of P62, P-PINK1 S228 , PINK1, P-Parkin S65 , Parkin, VDAC1, LC3B, and COXⅣ protein expression in primary mouse hepatocytes treated with HUA for different durations (0, 3, 6, 9, and 12 h). F – M , quantification of P62, P-PINK1 S228 , PINK1, P-Parkin S65 , Parkin, VDAC1, LC3B-II/I, and COXⅣ protein levels in primary hepatocytes (n = 3). N , representative WB images of P62, P-PINK1 S228 , and PINK1 protein levels in the mitochondrial fraction of HepG2 cells following HUA treatment at various time points (0, 3, 6, and 12 h). O – Q , quantification of mitochondrial P62, P-PINK1, and PINK1 protein levels in HepG2 cells (n = 3). R , representative WB images of UB protein expression in primary mouse hepatocytes treated with or without HUA and MG132 (10 μM) for 9 h. S , RT–PCR analysis of P62, PINK1, Parkin, MFN1, MFN2, OPA1, DRP1, and VDAC1 mRNA expression in primary hepatocytes treated with HUA for different durations (0, 3, 6, and 9 h). T , RT–PCR analysis of PGC1A, NRF1, and TFAM mRNA expression in primary hepatocytes treated with HUA for different durations (0, 6, 12, and 24 h). U , representative confocal microscopy images showing LC3B ( red ) and TOM20 ( green ) colocalization in primary hepatocytes treated with HUA for 9 h. Nuclei were counterstained with DAPI ( blue ). The scale bar represents 20 μm. V , quantification of LC3B mean fluorescence intensity (n = 3). W , Pearson’s correlation coefficient between LC3B and TOM20 from experiments shown in U (n = 3). Individual data points represent each independent experiment. Data are expressed as mean ± SD. Statistical analysis was done using unpaired t test and one-way ANOVA, Tukey’s post hoc analysis. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 compared with the indicated groups. DAPI, 4',6-diamidino-2-phenylindole.

Article Snippet: Antibodies for PINK1 (1:1000 dilution; catalog no.: 6946), P62 (1:1000 dilution; catalog no.: 5114), and F4/80 (1:200 dilution; catalog no.: 70076) were sourced from Cell Signaling Technology; P-Parkin S65 (1:1000 dilution; Ab315376 ) was from Abcam; P-PINK1 S228 (1:1000 dilution; AF7081) was from Affinity; TOM20 (1:200 dilution; catalog no.: 66777), Parkin (1:2000 dilution; catalog no.: 14060), ubiquitin (1:2000 dilution; catalog no.: 10201), and GAPDH (1:2000 dilution; catalog no.: 60004) from Proteintech, CD36 (1:1000dilution; NB400-144) from Novus Biologicals; and MFN2 (1:1000 dilution; A19678), LC3B (1:1000 dilution; A19665), and COXIV (1:1000 dilution; A11631) from Abclone.

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Confocal Microscopy, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Activation of mitophagy antagonizes high uric acid–induced hepatic lipid accumulation

doi: 10.1016/j.jbc.2025.111054

Figure Lengend Snippet: High uric acid induces mitophagy in mouse liver . A , representative Wesetrn blot (WB) images showing the expression of mitophagy-related proteins (P62, P-PINK1 S228 , PINK1, P-Parkin S65 , Parkin, VDAC1, and LC3B-II/I), mitochondrial fusion proteins MFN2 (mitofusin 2), and the mitochondrial marker COXIV in liver tissues from WT and Uox -KO mice. B – J , quantification of MFN2, P62, P-PINK1 S228 , PINK1, P-Parkin S65 , Parkin, VDAC1, LC3B-II/I, and COXIV protein levels (n = 3–5). K , RT–PCR analysis of mitochondrial dynamics–related gene expression in liver tissues from WT and Uox -KO mice (n = 4–8). L , gene set enrichment analysis (GSEA) plot for the “mitochondrial outer membrane” Gene Ontology term. Individual data points on the graph represent data from each mouse. Data are presented as mean ± SD. Statistical analysis was done using unpaired t test and one-way ANOVA, Tukey’s post hoc analysis. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 compared with the indicated groups.

Article Snippet: Antibodies for PINK1 (1:1000 dilution; catalog no.: 6946), P62 (1:1000 dilution; catalog no.: 5114), and F4/80 (1:200 dilution; catalog no.: 70076) were sourced from Cell Signaling Technology; P-Parkin S65 (1:1000 dilution; Ab315376 ) was from Abcam; P-PINK1 S228 (1:1000 dilution; AF7081) was from Affinity; TOM20 (1:200 dilution; catalog no.: 66777), Parkin (1:2000 dilution; catalog no.: 14060), ubiquitin (1:2000 dilution; catalog no.: 10201), and GAPDH (1:2000 dilution; catalog no.: 60004) from Proteintech, CD36 (1:1000dilution; NB400-144) from Novus Biologicals; and MFN2 (1:1000 dilution; A19678), LC3B (1:1000 dilution; A19665), and COXIV (1:1000 dilution; A11631) from Abclone.

Techniques: Expressing, Marker, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Activation of mitophagy antagonizes high uric acid–induced hepatic lipid accumulation

doi: 10.1016/j.jbc.2025.111054

Figure Lengend Snippet: Schematic diagram of activation of mitophagy antagonizes high uric acid–induced hepatic lipid accumulation (created with Figdraw.com ) . High uric acid upregulates CD36, promoting hepatic fat accumulation. Meanwhile, it activates mitophagy via the PINK1–Parkin pathway, which antagonizes this lipid accumulation.

Article Snippet: Antibodies for PINK1 (1:1000 dilution; catalog no.: 6946), P62 (1:1000 dilution; catalog no.: 5114), and F4/80 (1:200 dilution; catalog no.: 70076) were sourced from Cell Signaling Technology; P-Parkin S65 (1:1000 dilution; Ab315376 ) was from Abcam; P-PINK1 S228 (1:1000 dilution; AF7081) was from Affinity; TOM20 (1:200 dilution; catalog no.: 66777), Parkin (1:2000 dilution; catalog no.: 14060), ubiquitin (1:2000 dilution; catalog no.: 10201), and GAPDH (1:2000 dilution; catalog no.: 60004) from Proteintech, CD36 (1:1000dilution; NB400-144) from Novus Biologicals; and MFN2 (1:1000 dilution; A19678), LC3B (1:1000 dilution; A19665), and COXIV (1:1000 dilution; A11631) from Abclone.

Techniques: Activation Assay

Journal: Chinese Medicine

Article Title: Thonningianin A derived from Penthorum chinense Pursh alleviates cerebral ischemia/reperfusion-mediated apoptosis and pyroptosis through the activation of PINK1/Parkin-dependent mitophagy

doi: 10.1186/s13020-025-01247-2

Figure Lengend Snippet: TA activated mitophagy by PINK1/Parkin signaling pathway. A The Western blotting detection of the protein expressions of LC3I and LC3II in HT22 cells treated with TA and Rap (20 μM). B The bar chart presents the rate of LC3II/LC3I, * p < 0.05, * p < 0.01 and *** p < 0.01 versus the control group, n = 3. C Representative images of stable GFP-LC3 U87 cells treated with or without TA, Magnification: 20 × , scale bar: 100 μm. D The bar chart presents the rate of cells with GFP-LC3 puncta formation in stable GFP-LC3 U87 cells, * p < 0.05 and *** p < 0.001 versus control group. E Representative images of stable stable RFP-GFP-LC3 U87 cells treated with or without TA, Baf (100 nM) or Rap (10 μM), Magnification: × 64, scale bar: 5 μm. F Representative images of the co-localization of GFP-LC3 and MitoTracker in stable GFP-LC3 U87 cells with or without TA, CCCP or TA+AC220. G The bar chart indicates the rate of GFP/RFP, ** p < 0.05 and *** p < 0.001. H – K Line scan analysis showing the co-localization intensity (a.u.) of GFP-LC3 and MitoTracker in stable GFP-LC3 U87 cells under different treatments. L Representative images of HT22 cells transiently transfecting Mito-QC plasmid and treated with or without TA, CCCP or TA+AC220. M Representative images of BV2 cells transiently transfecting Mito-QC and treated with or without TA, CCCP or TA+AC220. N The bar chart indicates the rate of RFP/GFP in HT22 ransiently transfecting Mito-QC cells. ** p < 0.01 and *** p < 0.001 versus the control group, n = 3. O The bar chart indicates the rate of RFP/GFP in BV2 ransiently transfecting Mito-QC cells. ** p <0.01 and *** p < 0.001 versus the control group, n = 3. P Western blot detection of Parkin, p-Parkin, and PINK1 protein expression in HT22 cells treated with or without TA. Q , R The bar chart presents the rate of p-Parkin/Parkin (n = 3), PINK1/GAPDH (n = 5). ** p < 0.01 and *** p < 0.001 versus the control group

Article Snippet: Antibodies against GSDMD (20770-1-Ap), Caspase-3 (25158-1-Ap), Caspase-9 (10380-1-Ap), NQO1 (11451-1-Ap), GCLC (12601-1-Ap), LC3 (14600-1-Ap), PINK1 (23274-1-Ap), Parkin (14060-1-Ap), Antibody Caspase-1 (22,915-1-ap), GAPDH (6004-1-Ig), β-actin (66009-1-Ig), CoraLite ® Plus 488 goat anti-rabbit IgG (RGAR002), CoraLite ® Plus 594-Goat Anti-Mouse IgG (RGAR002), were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Western Blot, Control, Plasmid Preparation, Expressing

Journal: Chinese Medicine

Article Title: Thonningianin A derived from Penthorum chinense Pursh alleviates cerebral ischemia/reperfusion-mediated apoptosis and pyroptosis through the activation of PINK1/Parkin-dependent mitophagy

doi: 10.1186/s13020-025-01247-2

Figure Lengend Snippet: TA ameliorated neurological injury in MCAO/R Rats. A Representative immunofluorescence staining images of LC3 and NLRP3 in hippocampus of the brain in MCAO/R rats, × 10, scale bar: 100 μm. B The bar chart presents the counts of LC3 + cells, NLRP3 + /cells, and LC3 + /NLRP3 + co-localized cells. C – F The Western blotting detection of the protein expressions of LC3, PINK1, p-Parkin and NLRP3 in rat brain tissues across experimental groups. G – J The bar chart presents the ratios of LC3-II/I, PINK1/β-actin, p-Parkin/ Parkin, NLRP3/β-actin. * p < 0.05, ** p < 0.01 and *** p < 0.001versus the alone MCAO/R group, n = 3. K – N The Western blotting detection of the protein expressions of ASC, Caspase-1, IL-18 in rat brain tissues across experimental groups. O – R The bar chart presents the ratios of ASC/β-actin, Cleaved-Caspase-1/Pro- Caspase-1, Cleaved-IL-18/β-actin. * p < 0.05, ** p < 0.01 and *** p < 0.001versus the alone MCAO/R group, n = 3. S The Western blotting detection of the protein expression of IL-1β. T The bar chart presents the rate of Cleaved-IL-18/GAPDH. * p < 0.05 and ** p < 0.01 versus the alone MCAO/R group, n = 3. S The Western blotting detection of the protein expression of IL-1β. T The bar chart presents the rate of Cleaved-IL-1β/GAPDH. * p < 0.05 and ** p < 0.01 versus the alone MCAO/R group, n = 3. U The Western blotting detection of the protein expression of BAX and Bcl2. V The bar chart presents the rate of BAX/Bcl2. *** p < 0.001 versus the alone MCAO/R group, n = 3

Article Snippet: Antibodies against GSDMD (20770-1-Ap), Caspase-3 (25158-1-Ap), Caspase-9 (10380-1-Ap), NQO1 (11451-1-Ap), GCLC (12601-1-Ap), LC3 (14600-1-Ap), PINK1 (23274-1-Ap), Parkin (14060-1-Ap), Antibody Caspase-1 (22,915-1-ap), GAPDH (6004-1-Ig), β-actin (66009-1-Ig), CoraLite ® Plus 488 goat anti-rabbit IgG (RGAR002), CoraLite ® Plus 594-Goat Anti-Mouse IgG (RGAR002), were purchased from Proteintech Group, Inc (Wuhan, China).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing