Journal: Materials Futures

Article Title: A clinically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1088/2752-5724/ae4e4d

Figure Lengend Snippet: Substrate characterisation in 2D and 3D cell culture—biological responses and physical properties with different compositions. (a) hPSC confluence in 2D growth factor-reduced Matrigel (Geltrex), laminin 521, fibrin-laminin hydrogel, and fibrin gel after 4 d; scale bar = 100 μ m. (b) hPSCs cultured in 3D hydrogels. Top panel: fibrin (5 mg ml −1 ) gel. Bottom panel: Alphagel containing structures resembling pluripotent spheroids; scale bar = 200 μ m. Scanning electron microscopy of (c) fibrin gel and (d) Alphagel; scale bar = 1 μ m, magnification 20 K X, iProbe = 13 pA, 2.00 kV, Working Distancee = 4.4 mm for both images. (e) Young’s moduli in hydrogels with varying fibrin and laminin concentrations. One-way ANOVA; ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. (f) Cell viability of hPSCs cultured in Alphagel, fibrin-only hydrogels, and 2D standard substrates. Mean ± sandard error of the mean displayed. t -test; * = p < 0.05. (g) ELISA of laminin 521 in culture media used with acellular Alphagel and (h) the calculated amount of fibrin-bound laminin.

Article Snippet: Apolipoprotein B (APOB) secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, no. 3715-1H-6) according to the product literature.

Techniques: Cell Culture, Electron Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Materials Futures

Article Title: A clinically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1088/2752-5724/ae4e4d

Figure Lengend Snippet: Characterisation of iHeps derived in Alphagel, fibrin-only hydrogels, and Matrigel. (a) Key hepatocyte markers in Alphagel-derived iHeps. ALB = albumin, HNF = hepatocyte nuclear factor, CYP2A6 = Cytochrome P450 2A6, CD147 = cluster of differentiation protein 147, and E-CAD = E-cadherin. Scale bar = 25 μ m. (b) Key hepatocyte markers by gene expression (qPCR): CCAAT/enhancer-binding protein alpha (CEBPA), T-box transcription factor 3= TBX3, alpha-fetoprotein = AFP. (c) Albumin secretion (ELISA) and (d) CYP3A4 activity (P450-Glo TM ) in PHHs versus iHeps cultured in various gels (day 22). HCM = Hepatocyte Culture Media (Lonza). (e) LDL uptake (red) in Alphagel-derived iHeps versus hPSCs. Scale bar = 100 μ m. (f) CDFDA secretion (green) in Alphagel-derived iHeps versus hPSCs. Top panel scale bar = 20 μ m; bottom panel scale bar = 50 μ m. One-way ANOVA was used; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Article Snippet: Apolipoprotein B (APOB) secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, no. 3715-1H-6) according to the product literature.

Techniques: Derivative Assay, Gene Expression, Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Cell Culture

Journal: Materials Futures

Article Title: A clinically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1088/2752-5724/ae4e4d

Figure Lengend Snippet: Characterisation of iHeps cultured in Hepatogel and its effect on cell retention after intra-hepatic cell transplantation. (a) Differentially expressed genes in iHeps: Hepatologel, Alphagel, Matrigel, and adult PHHs. (b) A heat map summarising the differential gene expression of a hepatic 24-gene panel across replicates of Matrigel, Alphagel, and Hepatogel (normalised to hPSC). (c) Albumin ELISA of culture media and (d) luciferin-based measure of CYP3A4 activity: 2 d after completion of iHep differentiation and 2 d after plating PHHs. One-way ANOVA; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. (e) Mouse livers 3 d after intra-hepatic injection with H-iHeps in Hepatogel and 0.9% saline. Red box = area magnified. * = site of injection, dotted white lines demarcate engrafted cell mass. Scale bar = 1 mm. (f) Human albumin (stained red) in engrafted iHeps 3 d after intra-hepatic injection. Scale bar = 100 μ m. (g) H-iHeps identified by albumin staining on liver histology 3 d after intra-hepatic injection. (h) ELISA of mouse serum for human albumin after injection with H-iHeps in Hepatogel and 0.9% saline over time. Day 0 = serum levels before injection. T -test; *** = p < 0.001 and **** = p < 0.0001.

Article Snippet: Apolipoprotein B (APOB) secretion in the supernatant was quantified using the human APOB ELISA quantification kit (Mabtech, no. 3715-1H-6) according to the product literature.

Techniques: Cell Culture, Transplantation Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Activity Assay, Injection, Saline, Staining

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Full fabrication and application schematic diagram of GelMA-VEGF/ECM-PCSK9 composite hydrogel and the related signaling pathway of PCSK9 that promotes BMSC osteogenic differentiation.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques:

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Construction and characterization of GelMA-VEGF/ECM-PCSK9 composite hydrogel. A Schematic diagram showing the process of composite hydrogel construction; B) Photographs of GelMA-VEGF hydrogel and GelMA-VEGF/ECM-PCSK9 hydrogel formation after UV light respectively; C i) Electron microscopic image of pure GelMA hydrogel, with a scale of 100 μm; ii) Enlarged electron microscopic image of GelMA hydrogel, with a scale of 50 μm; D) i The electron microscope image of the combination of GelMA hydrogel and ECM, with a scale of 100 μm; ii Electron microscope magnified image of GelMA hydrogel combined with ECM, with a scale of 50 μm; E) The infrared spectrum (FITR) diagram of the acellular ECM, GelMA hydrogel and GelMA/ECM composite hydrogel contains common basic energy groups; F) Load rate of PCSK9 in ECM; G) Release rate of VEGF loaded with GelMA hydrogel and GelMA/ECM composite hydrogel respectively; H) Release rate of PCSK9 loaded with ECM and GelMA/ECM composite hydrogel respectively; I) Release rate of VEGF and PCSK9 loaded in GelMA and GelMA/ECM on different time points respectively; J) Release rate of VEGF and PCSK9 respectively when loaded in GelMA/ECM; K) The swelling rate of GelMA gel and GelMA/ECM composite gel dissolved in PBS (n = 6); L) Degradation rate of GelMA hydrogel and GelMA/ECM composite gel in vitro (n = 6).∗means that compared with the control group, p < 0.05; ∗means that compared with the control group, p < 0.01; ∗∗∗means that compared with the control group, p < 0.001.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: Microscopy, In Vitro, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Angiogenic capacity formulations of HUVECs in response to different composite biomaterial in vitro. A) Calcein/PI staining of HUVECs seeded on glass slides, showing the cell migration profiles of HUVECs treated with different material groups, scale bar = 200 μm; B) Quantitative analysis of the intercellular blank areas in each group, with the baseline group serving as the negative control; C) Angiogenic images of HUVECs co-cultured with different composite materials for 4 h and 8 h respectively, scale bar = 250 μm; D–G) Quantitative assessment of angiogenic capacity in each group via ImageJ software analysis of key angiogenic parameters. Abbreviations: NC = negative control group; V = exogenous VEGF protein-only group; GV=GelMA + exogenous VEGF protein group; GVE = GelMA + VEGF + ECM group; GVEP= GelMA/VEGF + ECM/PCSK9 group. Statistical notations: ∗∗means that compared with the control group, p < 0.01; ns = no significant difference between group.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: In Vitro, Staining, Migration, Negative Control, Cell Culture, Software, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: The effect of different composite hydrogel on the osteogenic differentiation of BMMSC in vitro. Cultivate BMMSC for osteogenic differentiation in osteogenic medium with GelMA, GelMA-VEGF, GelMA-VEGF/ECM, ECM-PCSK9, and GelMA-VEGF/ECM-PCSK9 for 7 days respectively. A,B) The cell nucleus was stained with DAPI (blue), RUNX2 was stained with RUNX2 antibody (green), and COL1A1 was stained with COL1A1 antibody (red), with a scale bar of 200 μm. C,D) The quantitative analysis results of COL1A1 and RUNX2 immunofluorescence images; E,F) Quantitative analysis of ALP staining and ARS staining for BMMSC co-culture with different kinds of hydrogels; G) ALP staining result for BMMSC co-culture with different kinds of hydrogels for 7days, scale bar = 200 μm; F) ARS staining result for BMMSC co-culture with different kinds of hydrogels for 14days, scale bar = 200 μm; I, J) After 7 and 14 days of co-culture with different combinations of composite hydrogels and BMMSC for osteogenesis and differentiation, the PCR experiment results of osteogenesis related indicators suggest that compared with the control group. G = simple GelMA hydrogel group, GV=GelMA hydrogels + VEGF protein group, GV/E = GelMA + VEGF/ECM group, EP = ECM + PCSK9 protein group, GVEP=GelMA + VEGF/ECM + PCSK9 protein group, the significant differences between the groups are expressed as ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns means there is no significant difference between the groups.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: In Vitro, Staining, Immunofluorescence, Co-Culture Assay, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: After adding different concentrations of PCSK9 to BMMSC for osteogenic induction, western blotting (WB) experiment was performed to evaluate the expression of phosphorylated proteins and total proteins among different osteogenic differentiation relevant signaling pathways. A) WB images of different signaling pathways that related to osteogenic differentiation after adding different concentrations of PCSK9; B-D) Quantitative analysis results of phosphorylated protein and total protein. Compared with the control group, ∗ means p < 0.05, ∗∗ means p < 0.01.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: Western Blot, Expressing, Protein-Protein interactions, Control

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: Human TNF-α Precoated ELISA Kit , Dakewe , 1117202.

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: Human TNF-α Precoated ELISA Kit , Dakewe , 1117202.

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 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: Human TNF-α Precoated ELISA Kit , Dakewe , 1117202.

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: Human TNF-α Precoated ELISA Kit , Dakewe , 1117202.

Techniques: Western Blot, Transfection, Expressing, Knockdown, Over Expression, Activity Assay, Concentration 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: Human IL-6 Precoated ELISA Kit , Dakewe , 1110602.

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: 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: Human IL-6 Precoated ELISA Kit , Dakewe , 1110602.

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: Human IL-6 Precoated ELISA Kit , Dakewe , 1110602.

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