Fig 1.
Temporal RNA-seq analysis of LPS-treated THP-1 macrophages.
(A) Workflow employed for RNA-Seq. (B) Statistics of differentially regulated transcripts in response to LPS stimulation (C) Volcano plots depicting differentially expressed genes in response to temporal LPS stimulation. The points colored red, blue, and black represent upregulated, downregulated, and unchanged transcripts, respectively. Temporal expression of TLR-induced transcripts including (D) MYD88 (E) TICAM1 (F) FOSB (G) JUNB (H) RELB (I) NFKB1, in THP-1 macrophages in response to LPS stimulation.
Fig 2.
Analysis of RNA-seq data from LPS stimulated macrophages reveals dysregulation of multiple signaling pathways.
(A) Heatstrip showing DEGs in response to temporal LPS stimulation (B) Gene set enrichment analysis revealed the enrichment of several pathways in response to LPS stimulation. Dotted lines highlight the dysregulation of gene clusters 1, 3, 4, and 7 belonging to the ‘response to lipopolysaccharide’ process. Temporal change of (C) IFNB1 and (D) CMPK2 transcripts in THP-1 macrophages in response to LPS stimulation.
Fig 3.
The expression pattern of CMPK2 is LPS and Poly (I:C) specific in THP-1 and mBMDMs.
The mRNA expression of (A) CMPK2 and (B) IFNβ in THP-1 cells standardized by 18s upon different TLR ligands: P3CSK4, FSL1, Poly (I:C), LPS K12, Flagellin, R837, R848, CL075, and CpG2006. The values are represented as the mean ± S.E. from three independent samples. (C) Western blot analysis of CMPK2 protein expression in THP-1 cells treated with LPS (200ng/ml), Poly (I:C) (10 μg/ml), and CLO75 (5 μg/ml) for the indicated time points. β-tubulin was used as the loading control. Quantitative real-time PCR of CMPK2 gene expression in BMDMs before and after (D) LPS and (F) Poly (I:C) challenge (n = 3 biological replicates pooled). Immunoblot analysis of CMPK2 in BMDMs after treatment with (E) LPS and (G) (Poly (I:C). Detection of β-actin was used as a loading control. One-way analysis of variance (ANOVA) with Dunnett post analysis was employed for the statistical analysis. (*p, 0.05, ***p, 0.001).
Fig 4.
CMPK2 is predominantly localized in the cytoplasm with partial mitochondrial overlap.
(A) THP-1 cells were subjected to confocal microscopy using anti-CMPK2, anti-TOM20 antibodies. Cell nuclei were stained with Hoechst. Scale bars: 5 μm. (B) Maximal projections and surface rendering of a z-stack is shown. Quantitative analysis of colocalization levels by Manders coefficient in THP-1 cells, analyzing the ratio between CMPK2 and TOM20. (C) The cytoplasmic (Cyt) and mitochondrial (Mito) fractions were prepared from THP-1 cells before and after 24 hours of LPS and Poly (I:C) stimulations and analyzed by Western blot for CMPK2. GAPDH and COX IV were used as markers for cytoplasmic proteins and mitochondrial inner membrane and intermembrane space proteins, respectively. Data are typical of three separate experiments.
Fig 5.
IFN-β treatment induces CMPK2 expression in THP-1 and BMDMs.
Quantitative RT-PCR of (A) CMPK2, (B) MX1, and (C) ISG15 in THP-1 cells stimulated with IFN-β (100 units/ml) for the indicated time. (D) Representative CMPK2, pStat1, and MX1 protein levels of THP-1 cells treated with IFN-β over a 0.25–9 hours time course. β-tubulin was used as a loading control. (E) Quantitative real-time PCR of CMPK2 and (F) Western blots of CMPK2 in mBMDMs, 2–6 hours after stimulation with IFN-β (10ng/ml) (n = 3 biological replicates pooled). β-actin was used as a loading control. Error bars represent the mean ± SEM of three independent experiments, and all figures are representative of three independent experiments. One-way analysis of variance (ANOVA), followed by Dunnett’s post hoc analysis, was employed for the statistical analysis. (**p, 0.01, ***p, 0.001).
Fig 6.
IFNAR and IRF3 are involved in the regulation of LPS and Poly (I:C)-mediated CMPK2 mRNA and protein expression.
THP-1 cells were treated with control mAbs (MOPC-173) or IFNAR chain 2 mAbs for 30 minutes, then stimulated with (A) LPS and (B) Poly (I:C). Lysates were used for Western blotting to detect CMPK2, p-Stat1, MX1, p-TBK1, and ISG15. β-tubulin was used as an equal loading control. (C) LPS and (E) Poly (I:C) treated wild-type, Irf3−/−, and Ifnar1−/− BMDMs mRNA expression of CMPK2 was measured by quantitative PCR. Immunoblot analysis of CMPK2 in lysates of wild-type, Irf3−/−, and Ifnar1−/− BMDMs after treatment with (D) LPS and (F) Poly (I:C). Error bars represent means ± SEM from three independent samples. All results were from three independent experiments.