Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Metabolic endotoxemia promotes neuroinflammation after focal cerebral ischemia

doi: 10.1177/0271678X19899577

Figure Lengend Snippet: Cellular localization of lipopolysaccharide (LPS), E. coli K99 and toll-like receptor 4 (TLR4) in the peri-infarct areas of ischemic brain. Double-immunofluorescence of (a) LPS, (b) E. coli K99, and (c) TLR4 in microglia/macrophages (Iba1), neurons (NeuN), endothelial cells (CD31), and astrocytes (GFAP) in the peri-infarct area 24 h after transient middle cerebral artery occlusion in db/db mice. High magnification images show that (a) LPS and (b) E. coli K99 were adherent on the surface of microglia/macrophages, neurons and endothelial cells (arrows). (c) TLR4 was colocalized with Iba1, NeuN and CD31 (arrows). Scale bars = 20 μm. A red square in the brain illustration shows the region of analysis. (d) The percentages of TLR4-immunoreactive areas in Iba1, NeuN, and CD31-positive cells in db/+, db/db, and polymyxin B (PL-B)-treated db/db mice (n = 6 per group). Data are shown as mean ± SD. **P < 0.01 and ***P < 0.001.

Article Snippet: 23 Double immunofluorescence staining was performed by simultaneous incubation of the sections with the following primary antibodies overnight at 4°C: anti- Escherichia coli LPS (1:100 dilution; Abcam) and anti- E. coli K99 (1:100 dilution; Lifespan, Providence, RI, USA), anti-TLR4 (1:100 dilution; Santa Cruz Biotechnology), anti-Iba1 (1:100 dilution; Abcam), anti-NeuN (neuron) (1:100 dilution; Abcam), anti-CD31 (endothelial cells) (1:25 dilution; Abcam), anti-glial fibrillary acidic protein (GFAP) (1:500 dilution; Abcam), and anti-transmembrane protein 119 (TMEM119) (1:150 dilution; Abcam).

Techniques: Immunofluorescence

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Bone marrow-derived macrophages (BMDMs) were obtained from wildtype or Cpeb4MKO mice. Immunoblot analysis of CPEB4 during lipopolysaccharide (LPS) stimulation. Vinculin served as loading control. ( B ) Percentage of mice of the indicated phenotype born from matings between Cpeb4 +/+ or Cpeb4 lox/lox females and a corresponding male carrying the Lyz2 Cre gene (n > 47 for each genotype). ( C ) Total animal body weight (n > 10). ( D ) Normalized organ weight (n = 6). ( E ) Complete blood counts from wildtype or Cpeb4MKO mice (n = 6). WBC, white blood cells; LYM, lymphocytes; MID, monocytes; GRA, granulocytes; PLT, platelets; RBC, red blood cells.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Western Blot, Control

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A–C ) Lipopolysaccharide (LPS)-stimulated wildtype (WT) bone marrow-derived macrophages (BMDMs). ( A ) Cpeb1–4 levels were measured by RT-qPCR (n = 6). ( B, left) CPEB4 immunoblot, using α-tubulin as loading control. (Right) CPEB4 quantification normalized to α-tubulin (n = 3; data shown in ). ( C , left) CPEB4 immunoblot in protein extracts treated with λphosphatase when indicated. (Right) Quantification of P-CPEB4 signal (n = 3). ( D–H ) LPS-stimulated WT and Cpeb4 –/– BMDMs. ( D ) Number of differentially expressed genes (p<0.01) between genotypes. mRNA levels were quantified by RNAseq (n = 4). Statistics: DESeq2 R package. ( E ) Z-score signature of the indicated pathways. mRNA levels were quantified by RNAseq (n = 4). Statistics: rotation gene set enrichment analysis. ( F , left) HIF1a and CPEB4 immunoblot, vinculin served as loading control. (Right) Normalized quantification, signal intensity was normalized to vinculin and fold change to WT at 9 hr after LPS induction was calculated (n = 3; data shown in ). ( G, H ) Hif1a and Il10 levels measured by RT-qPCR (n = 6). ( A, G ) Tbp was used to normalize. ( B, C, E, F ) Data are represented as mean ± SEM. ( F–H ) Statistics: two-way ANOVA. ( D, E ) See also . Figure 2—source data 1. Blots corresponding to and . Figure 2—source data 2. Blots corresponding to . Figure 2—source data 3. Blots corresponding to and .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Control

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Immunoblot analysis of CPEB4 in control or LPS-treated macrophages. α-Tubulin served as loading control. ( B ) Immunoblot analysis of CPEB4 in LPS-stimulated bone marrow-derived macrophages (BMDMs) obtained from wildtype or Cpeb4 –/– mice. Vinculin served as loading control.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Western Blot, Control, Derivative Assay

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Immunoblot analysis of HIF1a in LPS-stimulated bone marrow-derived macrophages (BMDMs) obtained from wildtype or Cpeb4 –/– mice. Vinculin served as loading control. Quantification is shown in . ( B ) Gene set analysis of Hallmark pathways in LPS-stimulated WT and Cpeb4 –/– BMDMs. Differential mRNA expression was measured by RNAseq (n = 4). Statistics: DESeq2 R package. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Western Blot, Derivative Assay, Control, Expressing

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysaccharide (LPS) and mRNA levels were measured by RT-qPCR, normalizing to Tbp (n = 6). Cpeb4 mRNA values are also shown in . ( B ) Schematic representation of the Cpeb4 3’-UTR showing AU-rich element (ARE) domains.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Cpeb4 levels in wildtype (WT) and p38αMKO bone marrow-derived macrophages (BMDMs) stimulated with lipopolysaccharide (LPS) (n = 3). ( B ) Cpeb4 levels in LPS-stimulated BMDMs treated with the p38α inhibitor PH-797804 (or DMSO as control) (n = 4). ( C ) WT or p38αMKO BMDMs were stimulated with LPS for 1 hr; Cpeb4 mRNA stability was measured after treating with actinomycin D (ActD). Statistics: paired t -test (60 min time point; n = 3). See also . ( D ) Cpeb4 mRNA levels in HuR RNA-immunoprecipitates (IP) performed in WT or p38αMKO BMDMs, after LPS stimulation as indicated. IgG IPs served as control. IP/input enrichment is shown, normalized to WT IP LPS (n = 2). See also . ( E ) Immunoblot of TTP protein in WT BMDMs treated with LPS for 0–9 hr. Vinculin served as loading control (n = 2). ( F ) Cpeb4 and Tnf decay rates in WT and TTPMKO BMDMs stimulated for 6 hr with LPS (data from ). Data represents the mean of three biological replicates. ( G, H ) U2OS cells were treated with tetracycline to induce the expression of a constitutively active MKK6, which induces p38α MAPK activation . ( G ) Cpeb4 levels upon p38α activation (+MKK6) or inhibition with SB203580 or PH-797804 (n = 3). ( H ) Cpeb4 levels in control or HuR-depleted U2OS cells, where p38α MAPK signaling has been activated (+MKK6) or inhibited (SB) (n = 2). See also . ( A–D, G, H ) mRNA levels were quantified by RT-qPCR. Gapdh ( A, B, C, G ) was used to normalize. ( A, B, D, G, H ) Data are represented as mean ± SEM. ( A, B ) Statistics: two-way ANOVA. ( C ) Statistics: paired t -test. ( G, H ) Statistics: one-way ANOVA, selected pvadj are shown. Figure 3—source data 1. Blots corresponding to .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Control, Western Blot, Expressing, Activation Assay, Inhibition, Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) WT or p38αMKO BMDMs were stimulated with lipopolysaccharide (LPS) for 1 hr; and mRNA stability was measured after treating with actinomycin D (ActD). mRNA levels were quantified by RT-qPCR. Gapdh was used to normalize.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) HuR IP was performed in wildtype (WT) and p38αMKO BMDMs stimulated with lipopolysaccharide (LPS) for 3 hr when indicated. IgG IP was used as control.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Control

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) TTP PAR-iCLIP was performed in bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS) for 6 hr. Coverage plots represent the number of crosslink sites (CL) detected in each position of Cpeb4 mRNA. For binding sites located in the Cpeb4 3′-UTR, distance to the transcription start site (TSS) and their scores is indicated (data from ).

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Binding Assay

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A–D ) CPEB4 RNA-Immunoprecipitation (IP) and sequencing was performed using total lysates (input) from wildtype (WT) or Cpeb4 –/– bone marrow-derived macrophages (BMDMs) that had been treated or not with LPS for 9 hr (n = 1). ( A ) CPEB4 immunoblot, using vinculin as a loading control. ( B ) Examples of read coverage of input or IP of selected mRNAs. Peak enrichments between WT and Cpeb4 –/– IPs are shown in blue. ( C ) Cytoplasmic polyadenylation element (CPE) and CPE G-containing transcripts according to ) in input and CPEB4 IPs. The script from was modified to consider TTTTGT as a CPE motif. Statistics: Fisher’s exact test. ( D ) Read coverage of IPs of selected mRNAs. ( E ) CPEB4 IP and RT-qPCR were performed for WT or Cpeb4 –/– BMDMs stimulated with LPS for 9 hr. IP/input enrichment is shown (n = 3). ( F ) Socs1 mRNA levels in LPS-stimulated WT and Cpeb4 –/– BMDMs. mRNA levels were measured by RT-qPCR normalizing to Tbp (n = 6). ( G ) Immunoblot of SOCS1 in WT and Cpeb4 –/– BMDMs treated with LPS. Vinculin served as loading control. Quantification is shown (FC to WT, after 9 hr of LPS) (n = 3). ( H ) Differential expression between WT and Cpeb4 –/– BMDMs treated with LPS measured by RNAseq (n = 4). Statistics: DESeq2 R package. ( I ) mRNA stability was measured by treating with actinomycin D (ActD) WT and Cpeb4 –/– BMDMs stimulated with LPS for the indicated times. Gene expression was analyzed by RT-qPCR, normalized to Gapdh/Tbp (n = 4). ( J ) RAW 264.7 macrophages were transfected with a Firefly luciferase reporter under the control of the cyclin B1 3′-UTR, containing either WT (CPE+) or mutated (CPE–) CPE motifs. The same plasmid contained Renilla luciferase reporter as a control. Macrophages were stimulated with LPS for 3 hr, at which point ActD was added. mRNA levels were measured by RT-qPCR. ( B, D ) Integrated Genomic Viewer (IGV) images. ( E–G ) Data are represented as mean ± SEM. ( F, G, I, J ) Statistics: two-way ANOVA. See also . Figure 4—source data 1. Blots corresponding to . Figure 4—source data 2. Blots corresponding to .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: RNA Immunoprecipitation, Sequencing, Derivative Assay, Western Blot, Control, Modification, Quantitative RT-PCR, Quantitative Proteomics, Gene Expression, Transfection, Luciferase, Plasmid Preparation

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A, B ) CPEB4 RNA-Immunoprecipitation (IP) and sequencing was performed in total lysates (Input) from wildtype and Cpeb4 KO bone marrow-derived macrophages (BMDMs), untreated or stimulated with LPS for 9 hr (n = 1). ( A ) CPE-A- or CPE-G-containing transcripts in Inputs and CPEB4 IPs. Statistics with Fisher’s exact test. ( B ) Top 10 Gene Ontology KEGG categories enriched in CPEB4 target mRNAs in wildtype BMDMs stimulated with LPS for 9 hr. Mus musculus transcriptome was used as background. Statistics: Benjamini–Hochberg adjusted p-value is shown. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: RNA Immunoprecipitation, Sequencing, Derivative Assay

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A, B ) Differential expression between wildtype (WT) and bone marrow-derived macrophages (BMDMs) treated with LPS measured by RNAseq (n = 4). Statistics: DESeq2cR package ( C ) Dusp1 mRNA levels were measured by RT-qPCR, normalizing to Tbp .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Quantitative Proteomics, Derivative Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) ARE-containing transcripts in the input and CPEB4 immunoprecipitations (IPs) from . Statistics: Fisher’s exact test. ( B ) Percentage of CPEB4 targets in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) transcriptome and TTP targets in LPS-stimulated BMDMs . ( C ) Genome-wide correlation between ARE and CPE motifs in 3′-UTRs. The black line shows the linear regression trend line. R 2 = 0.6364. ( D ) mRNA levels in wildtype (WT) and Cpeb4 –/– BMDMs were measured by RT-qPCR, normalizing to Tbp (n = 6). Statistics: two-way ANOVA. Socs1 data are also shown in . ( E ) mRNA expression in WT or TTPMKO BMDMs treated with LPS. Statistics: DESeq2 software, qval is shown (data from ). ( F ) Common TTP and CPEB4 target mRNAs were classified according to the ARE:CPE score as ARE-dominant (ARE-d; red; 30 mRNAs) or CPE-dominant (CPE-d; blue; 61 mRNAs) (see also ). ( G ) CPEB4 and TTP target mRNAs were plotted according to the number of AREs and CPEs in the 3′-UTR. (Left) The dashed line separates ARE-d and CPE-d mRNAs. (Right) mRNAs were classified according to only the number of AREs in their 3'-UTR. The dashed line separates ARE high (>4 AREs; yellow; 43 mRNAs) from ARE low (≤4 AREs; navy; 56 mRNAs) mRNAs. ( H, I ) WT BMDMs were stimulated with LPS and mRNA levels were quantified by RNAseq (n = 4). ( H ) Common CPEB4 and TTP target mRNAs were classified as AREd/CPEd (left) or ARE high /ARE low (right). For each mRNA, the levels after 9 hr of LPS treatment were normalized by its expression at 6 hr LPS. ( I ) 1521 CPE- and ARE-containing mRNAs were classified as sustained >0.5 (1319 mRNAs) or downregulated <0.5 (202 mRNAs) according to their expression after 9 hr of LPS treatment, after normalizing to the peak of expression throughout the LPS response. For each mRNA, the ARE:CPE score was calculated. ( J, K ) RAW 264.7 macrophages were transfected with a Firefly luciferase reporter under the control of a chimeric 3′-UTR combining Ier3 and cyclin B1 AREs and CPEs motifs, respectively. Inactivating specific CPE or ARE motifs, six different 3′-UTRs with distinct ARE:CPE scores were generated. The same plasmid contained Renilla luciferase reporter as a control. ( J ) Scheme of the six constructs used for the dual luciferase reporter assay. Inactivated motifs are shown in gray. ( K ) RAW 264.7 macrophages were stimulated with LPS for 6 hr and Firefly/Renilla levels were measured by RT-qPCR. Values were normalized to the 0AREs/0CPEs construct. Statistics: one-way ANOVA Friedman test. All significant differences are shown except 5AREs/0CPEs vs. 0AREs/3CPEs (**) and 2AREs/3CPEs (*). ( D, H, K ) Data are represented as mean ± SEM. ( H, I ) Statistics: Mann–Whitney t -test. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Genome Wide, Quantitative RT-PCR, Expressing, Software, Transfection, Luciferase, Control, Generated, Plasmid Preparation, Construct, Reporter Assay, MANN-WHITNEY

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) ARE/CPE score definition. ARE and CPE motifs used to calculate the score are specified. ( B–E ) Wildtype bone marrow-derived macrophages (BMDMs) were treated with lipopolysaccharide (LPS) and mRNA levels were quantified by RNAseq (n = 4). ( B–D ) Common TTP and CPEB4 target mRNAs were considered. ( B ) ARE-dominant (AREd, red) and CPE-dominant (CPEd, blue) mRNA levels after 3 hr of LPS stimulation, normalized for its expression at 1 hr. Statistics: Mann–Whitney t -test. ( C ) Mean expression profile of CPEd and AREd mRNAs in LPS-stimulated BMDMs. For each mRNA, values were normalized to its peak of expression. Statistics: two-way ANOVA. ( D ) Mean mRNA expression profile of ARE high and ARE low mRNAs in LPS-stimulated BMDMs. For each mRNA, values were normalized to its peak of expression. ( E ) 1521 CPE- and ARE-containing mRNAs were classified as sustained >0.5 or downregulated <0.5, on the basis of their expression after 9 hr of LPS treatment, normalized by their peak of expression during the LPS response. For each mRNA, the number of AREs in its 3’-UTR was calculated. ( B ) Data are represented as mean ± SEM. ( C, D ) Data are represented as mean ± SD. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Lipopolysaccharide (LPS) stimulates the MAPK signaling cascades downstream of TLR4. p38α controls TTP phosphorylation, causing a shift in the competitive binding equilibrium between Hu-antigen R (HuR) and tristetraprolin (TTP) towards HuR, which stabilizes AU-rich element (ARE)-containing mRNAs. The p38α/HuR/TTP axis also regulates Cpeb4 mRNA stability and promotes CPEB4 expression during the late phase of the LPS response. CPEB4 then accumulates in its active state, which involves phosphorylation by ERK1/2 MAPK signaling. During the resolutive phase of the LPS response, CPEB4 and TTP compete to stabilize/destabilize mRNAs containing cytoplasmic polyadenylation elements (CPEs) and AREs. The equilibrium between these positive and negative cis -acting elements in the mRNA 3′-UTRs generates customized temporal expression profiles. CPEB4 stabilizes CPE-dominant mRNAs, which are enriched in transcripts encoding negative regulators of MAPKs that contribute to inflammation resolution. ( B ) Immunoblot of the indicated proteins in wildtype (WT) BMDMs treated with LPS for 0–9 hr. Vinculin and Ponceau staining served as loading control. Figure 6—source data 1. Blots corresponding to .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Western Blot, Staining, 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: Mice in the LPS+3-HPA group were intraperitoneally injected with 3-hydroxypropanoic acid (3-HPA, Macklin) at a dose of 50 mg/kg 2 h before LPS administration; mice in the control group were injected with an equal volume of physiological saline.

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: Mice in the LPS+3-HPA group were intraperitoneally injected with 3-hydroxypropanoic acid (3-HPA, Macklin) at a dose of 50 mg/kg 2 h before LPS administration; mice in the control group were injected with an equal volume of physiological saline.

Techniques: Expressing, Gene Expression