Journal: Animal Cells and Systems

Article Title: Pharmacological inhibition of REV-ERBα ameliorates motor deficits in the 6-OHDA-lesioned mouse model of Parkinson’s disease

doi: 10.1080/19768354.2026.2690728

Figure Lengend Snippet: SR8278 treatment does not restore the expression of TH or other DAergic markers in the SN of 6-OHDA-lesioned mice. (A) Representative western blot images of TH in the SN from sham- and 6-OHDA-lesioned mice treated with VEH or SR8278. β-actin was used as a loading control. Three independent sets are shown. (B) Densitometric quantification of TH protein levels normalized to β-actin. (C-E) Quantitative RT-PCR analysis of VMAT2, DAT, and DRD2 mRNA expression in the SN from sham- and 6-OHDA-lesioned mice treated with VEH or SR8278. For all experiments shown in this figure, SR8278 was administered 3 h prior to tissue collection, and SN tissues were collected at CT22–01. Data are presented as mean ± SEM, with individual values shown (n = 3 for B; n = 3–4 for C–E). Statistical significance was determined by two-way ANOVA followed by Tukey’s post hoc analysis. ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were blocked in Tris-buffered saline (TBS; 10 mM Tris [pH 7.6], 150 mM NaCl, 2 mM MgCl2) supplemented with 0.3% Tween-20 and 5% bovine serum albumin (BSA) and incubated with primary antibodies against tyrosine hydroxylase (TH; T2928, Sigma-Aldrich) and β-actin (sc-47778, Santa Cruz Biotechnology) at room temperature for 1 h. After washing four times with TBS containing 0.3% Tween-20, membranes were incubated with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Analysis, Tissue

Journal: Journal of Cellular and Molecular Medicine

Article Title: Arachidonic Acid Disrupts PKM2 ‐Linked Glycolysis‐Related Metabolism and Exacerbates PAN ‐Induced Podocyte Injury

doi: 10.1111/jcmm.71404

Figure Lengend Snippet: Aa aggravated PAN‐induced podocyte injury in vitro. Control podocytes were treated with PBS. PAN‐treated podocytes received 250 μM PAN for 24 h. PAN+AA‐treated podocytes received PAN (250 μM) and AA (5 × 10 −7 M) for 24 h. AA‐treated podocytes received AA (5 × 10 −7 M) for 24 h. (A) Representative phalloidin staining images of F‐Actin (green) and nuclei (blue), with quantification of F‐Actin mean intensity per cell ( n = 3). Scale bar, 20 μm. (B) Western blot analysis and quantification of nephrin protein expression in cultured podocytes ( n = 6). Data are expressed as the mean ± SD and analysed by one‐way ANOVA with Bonferroni correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against β‐actin (1:2000, Sigma‐Aldrich, MO, USA), nephrin (1:500, PROGEN), PKM1 (1:1000, Abcam), PKM2 (1:1000, Proteintech), phospho‐PKM2 (Tyr105 1:1000, Proteintech), β‐actin (1:1000, CST), and TOMM20 (1:1000, CST).

Techniques: In Vitro, Control, Staining, Western Blot, Analysis, Expressing, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Arachidonic Acid Disrupts PKM2 ‐Linked Glycolysis‐Related Metabolism and Exacerbates PAN ‐Induced Podocyte Injury

doi: 10.1111/jcmm.71404

Figure Lengend Snippet: PKM2 Activation attenuated PAN + AA‐induced podocyte injury and mitochondrial dysfunction in vitro. PAN+AA‐treated podocytes were exposed to PAN (250 μM) and AA (5 × 10 −7 M) for 24 h. PAN+AA+TEPP‐46‐treated podocytes were exposed to TEPP‐46 (10 μM) under PAN+AA conditions. T denotes TEPP‐46 in the figure. (A) Representative phalloidin staining images of F‐Actin and quantification of F‐Actin mean intensity per cell ( n = 3). Scale bar, 20 μm. (B) Representative TUNEL staining images and quantification of apoptotic podocytes ( n = 3). Scale bar, 50 μm. (C, D) Representative immunofluorescence images and quantification of PKM2 and phospho‐PKM2. Nephrin is shown in red, PKM2 or phospho‐PKM2 in green, and DAPI in blue ( n = 3). Scale bar, 20 μm. (E) Western blot analysis and quantification of PKM2 protein expression ( n = 6). (F) Western blot analysis and quantification of phospho‐PKM2 (p‐PKM2) protein expression ( n = 6). (G) Western blot analysis and quantification of TOM20 protein expression ( n = 6). (H) Representative immunofluorescence images and quantification of TOM20 ( n = 3). Scale bar, 20 μm. (I) Transmission electron microscopy images and quantification of mean mitochondrial area, cristae‐damage rate and mitochondrial damage score ( n = 3). Scale bar, 2.0 μm. (J) MitoTracker staining and quantification of mitochondrial fluorescence intensity ( n = 3). Scale bar, 20 μm. (K) JC‐1 staining and quantification of mitochondrial membrane potential ( n = 3). Scale bar, 20 μm. (L) Representative DCFH‐DA staining images and quantification of ROS levels ( n = 3). Scale bar, 20 μm. (M) PK activity, lactate production, pyruvate production and ATP levels ( n = 3). Data are expressed as mean ± SD. Statistical significance was determined by unpaired Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against β‐actin (1:2000, Sigma‐Aldrich, MO, USA), nephrin (1:500, PROGEN), PKM1 (1:1000, Abcam), PKM2 (1:1000, Proteintech), phospho‐PKM2 (Tyr105 1:1000, Proteintech), β‐actin (1:1000, CST), and TOMM20 (1:1000, CST).

Techniques: Activation Assay, In Vitro, Staining, TUNEL Assay, Immunofluorescence, Western Blot, Analysis, Expressing, Transmission Assay, Electron Microscopy, Fluorescence, Membrane, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Arachidonic Acid Disrupts PKM2 ‐Linked Glycolysis‐Related Metabolism and Exacerbates PAN ‐Induced Podocyte Injury

doi: 10.1111/jcmm.71404

Figure Lengend Snippet: Proposed working model of AA‐associated aggravation of PAN‐induced podocyte injury. Based on our previous study, AA may activate the PKA‐c‐Abl‐nephrin axis and contribute to nephrin phosphorylation and actin cytoskeletal remodelling. In the present study, PAN+AA was associated with reduced PKM2 abundance, increased PKM2 Tyr105 phosphorylation, decreased PK activity, lower pyruvate/lactate/ATP levels, mitochondrial injury, ROS accumulation and apoptosis. These cytoskeletal and metabolic stress pathways may converge to aggravate podocyte injury and proteinuria. Potential crosstalk between these pathways is proposed, but the sequence and causal relationship remain to be experimentally validated.

Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against β‐actin (1:2000, Sigma‐Aldrich, MO, USA), nephrin (1:500, PROGEN), PKM1 (1:1000, Abcam), PKM2 (1:1000, Proteintech), phospho‐PKM2 (Tyr105 1:1000, Proteintech), β‐actin (1:1000, CST), and TOMM20 (1:1000, CST).

Techniques: Phospho-proteomics, Cytoskeletal, Activity Assay, Sequencing