tsg101  (Proteintech)

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA inhibits the secretion of inflammatory factors and glycolysis in macrophages (A and B) Schematic diagram of THP-1 cell (A) and BMDMs (B) activation into pro-inflammatory macrophages. Created with BioRender.com. (C) The concentrations of IL-6, TNF-α, and IL-1β in THP-1 cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (D) The concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with different concentrations of 3-HPA (0, 0.625, 1.25, 5 mM) followed by LPS stimulation. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (E) The concentrations of IL-6, TNF-α, and IL-1β in BMDM cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL) or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Bubble plot of KEGG pathway enrichment analysis for differentially expressed genes between LPS (100 ng/mL) and LPS+3-HPA (5 mM) treated in THP-1 cells. The size of each bubble represents the number of differentially expressed genes, and the color indicates the enrichment factor. (G and H) Pyruvate and lactate levels in THP-1 cells (G) and BMDMs (H) treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM). (I) Immunoblots of protein expression levels of HK, GAPDH, PKM, and LDHA in THP-1 cells treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM), and quantitative results of GAPDH. (J) The mRNA levels of GAPDH in THP-1 cells treated with LPS (100 ng/mL) or LPS+3-HPA (5 mM). (K) GAPDH activity assay in THP-1 cells and BMDMs treated with PBS, LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data in (G–K) are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Activity Assay

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH is modified with carboxyethylation at Cys 247 (A) Mass spectrometry analysis of GAPDH peptide (234–260) with carboxyethylation and 3-HPA (5 mM) incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. (B) SPR analysis of the affinity of the anti-ceC247 antibody for the carboxyethylation modified GAPDH peptide (234–260) and unmodified GAPDH peptide (234–260). (C) ELISA-based binding curve of anti-ceC247 antibody to modified GAPDH peptide (GAPDH ce (234–260)) and unmodified GAPDH peptide (234–260). Data are the means ± SD and n = 3 per group. Statistical significance was determined using two-way ANOVA followed by ∗∗p < 0.01. (D) Chemical structures of cysteine carboxyethylation and cysteine lactylation. (E) Unmodified GAPDH peptide (234–260), carboxyethylated peptide (GAPDH ce (234–260)), and lactylated peptide (GAPDH lac (234–260)) were tested with the anti-ceC247 antibody in dot blot assays. (F) Immunoblots of lysates from 293 T cells overexpressing GAPDH, which were treated with 5 mM 3-HPA and 5 μM MG132. The blots were probed with the anti-ceC247 antibody. (G) 3-HPA incubated with the GAPDH peptide (234–260) at 37 °C for 4 h. An anti-ceC247 antibody and the anti-wtC247 antibody were used in dot blot assays.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Modification, Mass Spectrometry, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Dot Blot, Western Blot

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA-induced carboxyethylation of GAPDH promotes its degradation through the ubiquitin-proteasome pathway (A) Chemical structure of carboxyethylated cysteine (left) and structures of aspartic acid (D), glutamic acid (E), methionine (M), and cysteine (C). (B) Immunoblot of GAPDH after transient transfection of flag-tagged GAPDH(C), GAPDH(D), GAPDH(M), GAPDH(E) plasmid in 293 T cells at 24 h, 36 h, and 48 h. (C) Immunoblot of GAPDH after CHX treatment. The GAPDH antibody was used to compare the degradation rates of GAPDH(M), GAPDH(E), GAPDH(D), and GAPDH(C). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ns, not significant. (D) Immunoblot and quantitative analysis of GAPDH ce after CHX treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the degradation rates of carboxyethylated GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ns, not significant. (E) Immunoblot and quantitative analysis of GAPDH ce after 3-HPA treatment. The anti-ceC247 antibody and anti-GAPDH antibody were used to compare the content of carboxyethylated GAPDH and total GAPDH. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05; ∗∗p < 0.01; ns, not significant. (F) Immunoblot and quantitative analysis of GAPDH ce in 293 T cells treated with 3-HPA (5 mM) combined with proteasomal inhibitor MG132, autophagic inhibitor Chloroquine, or lysosomal inhibitor Bafilomycin A1. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01; ∗∗∗∗p < 0.0001; ns, not significant. (G) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub. Cells were treated with 3-HPA (5 mM) and MG132 (5 μM) for 24 h. (H) Immunoprecipitation of GAPDH or GAPDH ce followed by immunoblotting for Myc in 293 T cells transfected with Myc-Ub mutants (K6O, K11O, K27O, K29O, K33O, K48O, K63O). Cells were treated with 3-HPA (5 mM) for 24 h.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA enhances the metabolite content of the TCA cycle and mitochondrial oxidation (A) Schematic diagram of THP-1 with 2-DG treatment. Created with BioRender.com. (B) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS, LPS+3-HPA, LPS+2-DG, or LPS+3-HPA+2-DG. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (C) Schematic diagram of THP-1 with high glucose treatment. Created with BioRender.com. (D) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS+3-HPA, LPS+3-HPA+glucose. Data are the means ± SD and n = 3 per group. Statistical significance was determined using unpaired Student’s t test with ∗∗∗p < 0.001; ns, not significant. (E) KEGG pathway enrichment analysis of metabolic pathways in BMDM cells treated with LPS or LPS+3-HPA. (F) Relative abundance of metabolite (ornithine, citrulline, L-malate, succinic acid, trans-aconitic acid, cis-aconitic acid) in BMDM cells treated with LPS or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using unpaired Student’s t test with ∗p < 0.05; ∗∗p < 0.01. (G) Correlation network of metabolites and genes in the metabolic pathway. Nodes represent metabolites (blue squares) and genes (colored circles). Gray edges indicate pairwise correlations between metabolites and genes. The colors similarly represent expression levels, with red typically indicating higher expression and blue indicating lower expression compared to the mean. (H) Schematic diagram of arginine metabolism and the TCA cycle. (I) Heatmap of mitochondrial oxidation-related gene expression associated with differentially expressed metabolites. Red indicates relatively high gene expression, while blue indicates relatively low gene expression within each row. (J) Schematic diagram of the catalytic function of the GAPDH enzyme. (K) Concentrations of the NAD + /NADH ratio in THP-1 cells and BMDM cells treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; (L) ATP concentrations in THP-1 cells and BMDMs treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05; ∗∗∗∗ p < 0.0001; ns, not significant. (M) Representative images and mitochondrial analysis of BMDM cells treated with PBS, LPS, or LPS+3-HPA. Scale bars, 1 μm (upper) and 0.25 μm (lower). For mitochondrial number, n = 8–12 ( n = 12 for PBS, n = 8 for LPS, n = 9 for LPS+3-HPA group).

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Expressing, Gene Expression

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH carboxyethylation inhibited macrophage glycolysis and the release of inflammatory factors (A) Schematic workflow illustrating the strategy of silencing endogenous GAPDH via 3′UTR-targeting siRNA and overexpressing exogenous GAPDH. (B) Immunoblot and quantitative analysis of GAPDH protein in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01. (C) Relative mRNA expression of GAPDH in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗∗∗p < 0.0001. (D) Immunoblot analysis of FLAG-tagged exogenous GAPDH(E) and GAPDH in 293 T cells. Knockdown of endogenous GAPDH with siRNA followed by the overexpression of GAPDH (E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test with ∗p < 0.05; ns, not significant. (E) Relative mRNA expression of GAPDH in 293 T cells knockdowned endogenous GAPDH (siGAPDH) and overexpressed GAPDH (C) and GAPDH (E), respectively. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (F) GAPDH activity assay in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗∗∗p < 0.0001; ns, not significant. (G) Concentrations of lactate and pyruvate in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (H) Relative mRNA expression of IL-6 , TNF-α , and IL-1β in THP-1 cells which transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (I) The concentration of TNF-α in THP-1 cells that overexpressed GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Subsequently, cells were incubated overnight at 4°C with primary antibodies: rabbit anti-GAPDH polyclonal antibody (R1210-1, HUABIO, 1:200 dilution) and mouse anti-ceC247 monoclonal antibody (1:200 dilution).

Techniques: Western Blot, Transfection, Expressing, Knockdown, Over Expression, Activity Assay, Concentration Assay