Journal: Cell Reports Medicine

Article Title: Artificial exosomes synergistically reshape sepsis immune homeostasis by modulating neutrophil fate and blocking PD-1/PD-L1

doi: 10.1016/j.xcrm.2026.102819

Figure Lengend Snippet: Preparation, characterization, and interaction of AT@NV-PD1 nanoparticles with neutrophils (A) CLSM images show PD-1 (green) expression on engineered RAW 264.7 membranes (scale bars, 15 μm). (B and C) Flow cytometry quantifies PD-1 expression in primary vs. engineered cells. (D) Western blot analysis of PD-1 expression in primary and genetically engineered RAW 264.7 cells. (E and F) DLS and TEM reveal AT@NV-PD1’s hydrodynamic diameter (110–130 nm) and core-shell structure (scale bars, 50 nm). (G) CLSM demonstrates AT@NV-PD1 (red) uptake by activated neutrophils (scale bars, 10 μm). (H) Anti-PD-L1 pretreatment blocks uptake (scale bars, 10 μm). (I) Resting neutrophils show minimal uptake (scale bars, 10 μm). (J) Flow cytometry shows elevated PD-L1 on septic neutrophils. (K and L) Enhanced AT@NV-PD1 uptake in CLP-model neutrophils vs. healthy controls. (M–Q) Nanodecoys effectively neutralize inflammatory mediators (TNF-α, IL-1β, IL-6, PD-L1, LPS). All data are presented as mean ± SD ( n = 3), ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns, not significant.

Article Snippet: Lipopolysaccharide (LPS) , MedChemExpress (USA) , Cat. HY-D1056A1.

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Cell Reports Medicine

Article Title: Artificial exosomes synergistically reshape sepsis immune homeostasis by modulating neutrophil fate and blocking PD-1/PD-L1

doi: 10.1016/j.xcrm.2026.102819

Figure Lengend Snippet: AT@NV-PD1 regulates neutrophil fate and its mechanism in vitro (A and B) Flow cytometry analysis and quantitative results showing that AT@NV-PD1 treatment can significantly induce apoptosis in neutrophils stimulated by IFN-γ and LPS. (C) Representative immunofluorescence images of NETs; CitH3 (red), MPO (green), and DAPI (blue) staining, (scale bars, 20 μm). (D) Representative CLSM images of SYTOX Green-stained NETs. Nuclear DNA was stained with DAPI (blue), and extracellular DNA was stained with SYTOX Green (green), (scale bars, 20 μm). (E and F) Expression levels of NETs and IL-6 in neutrophils treated with different formulations, as measured by ELISA. Data are presented as mean ± standard deviation. Differences were evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test, n = 5, ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗ p < 0.0001.

Article Snippet: Lipopolysaccharide (LPS) , MedChemExpress (USA) , Cat. HY-D1056A1.

Techniques: In Vitro, Flow Cytometry, Immunofluorescence, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cell Reports Medicine

Article Title: Artificial exosomes synergistically reshape sepsis immune homeostasis by modulating neutrophil fate and blocking PD-1/PD-L1

doi: 10.1016/j.xcrm.2026.102819

Figure Lengend Snippet: AT@NV-PD1 attenuates T cell exhaustion and promotes immune function recovery and does not impair neutrophil generation in the bone marrow (A) Flow cytometry analysis of the CD4 + /CD8 + T cell ratio in peripheral blood from CLP mice 5 days after intervention with different formulations. (B and C) Representative flow cytometry plots (B) and quantitative analysis (C) of CD4 + T cells in peripheral blood. (D and E) Representative images (D) and quantification (E) of TUNEL staining for apoptosis in splenocytes from healthy control and sepsis-treated mice (scale bars, 100 μm). (F and G) Representative flow cytometry plots (F) and quantitative analysis (G) of Treg cells in peripheral blood. (H) Flow cytometry plots of CD11b + cells in peripheral blood. (I and J) Representative flow cytometry plots and quantitative analysis of M-MDSC cells in peripheral blood. (K) Mouse body weights were monitored during the experiment, including healthy mice, PBS-treated sepsis mice, and AT@NV-PD1 -treated sepsis mice (AT7519 at a dose of 5 mg/kg). (L–O) Mice were subjected to intraperitoneal (i.p.) LPS injection to establish a sepsis model, and AT@NV-PD1 treatment was administered 4 h after LPS administration. Four hours later, LPS-challenged mice were intravenously (i.v.) injected with AT@NV-PD1 (AT7519 at a dose of 5 mg/kg). Control mice received neither LPS nor AT@NV-PD1 treatment. After 72 h, all surviving mice and control (healthy) mice were challenged with LPS (i.t. [intratracheally], 10 mg/kg). At 84 h, bronchoalveolar lavage fluid (BALF) was collected to assess neutrophil counts, IL-1β, TNF-α, and IL-6 levels. All data are presented as mean ± standard deviation (SD), and differences were evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test ( n = 5; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001).

Article Snippet: Lipopolysaccharide (LPS) , MedChemExpress (USA) , Cat. HY-D1056A1.

Techniques: Flow Cytometry, TUNEL Assay, Staining, Control, Injection, Standard Deviation

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