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. 2022 Apr 20;11:e75873. doi: 10.7554/eLife.75873

Figure 2. Inflammation resolution is impaired in Cpeb4–/– macrophages.

(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 Figure 2—figure supplement 2). (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 Figure 2—figure supplement 2). (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 Supplementary file 1.

Figure 2—source data 1. Blots corresponding to Figure 2B and Figure 2—figure supplement 1A.
Figure 2—source data 2. Blots corresponding to Figure 2C.
Figure 2—source data 3. Blots corresponding to Figure 2F and Figure 2—figure supplement 3A.

Figure 2.

Figure 2—figure supplement 1. CPEB4 upregulation in lipopolysaccharide (LPS)-stimulated macrophages.

Figure 2—figure supplement 1.

(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.
Figure 2—figure supplement 2. Differentiation of Cpeb4–/– bone marrow derived macrophages.

Figure 2—figure supplement 2.

(A) Mean expression of myeloid markers in wildtype and Cpeb4–/– bone marrow-derived macrophages (BMDMs) was analyzed by flow cytometry. For each experiment, fold change to wildtype was calculated (n = 10).
Figure 2—figure supplement 3. Characterization of lipopolysaccharide (LPS) response in Cpeb4–/– macrophages.

Figure 2—figure supplement 3.

(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 Figure 2E. (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 Supplementary file 1.