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

Figure 4. CPEB4 stabilizes mRNAs encoding negative feedback regulators of the lipopolysaccharide (LPS) response.

(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 Piqué et al., 2008) in input and CPEB4 IPs. The script from Piqué et al., 2008 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 Supplementary files 1-2.

Figure 4—source data 1. Blots corresponding to Figure 4A.
Figure 4—source data 2. Blots corresponding to Figure 4G.

Figure 4.

Figure 4—figure supplement 1. Characterization of CPEB4 targets in lipopolysaccharide (LPS)-stimulated macrophages.

Figure 4—figure supplement 1.

(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 Supplementary file 2.
Figure 4—figure supplement 2. Expression of negative feedback regulators of the lipopolysaccharide (LPS) response is impared in Cpeb4–/– bone marrow-derived macrophages (BMDMs).

Figure 4—figure supplement 2.

(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.