Fig 1: Effects of CMPK2 KO on the DENV infection-induced release of mtDNA and activation of TLR9(A−G) THP-1 cells and THP-1 CMPK2-KO clones were infected with DENV (MOI = 5), and the levels of total mtDNA and cytosolic mtDNA were measured by qPCR (A). Both 16S levels and ND5 levels were normalized to nuclear DNA (actin or albumin as indicated) or the exogenously introduced FLAG level. BMDCs treated with control siRNA or CMPK2-specific siRNA were infected with DENV (MOI = 1), and the levels of total and cytosolic mtDNA were measured by qPCR (B). Both 12S levels and D-loop levels were normalized to nuclear DNA (B2M or TERT) or the exogenously introduced FLAG level. The intensity of TLR9 expression in BMDCs treated with CMPK2-specific siRNA or control siRNA was determined by flow cytometry (C). The mRNA expression of TLR9 in wild-type cells and CMPK2-KO clones was determined by qPCR (D). The knockdown of CMPK2 expression in BMDCs also resulted in a reduction in the DENV infection-induced expression of 8-OHdG measured by flow cytometry (E) and confocal microscopy (F). Scale bar, 5 μm. Similarly, knockdown of CMPK2 expression reduced DENV-induced 8-OHdG expression in human DCs (G). Values represent the mean of the individual measurements in each sample ±SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. p value was calculated by the Student's t test (A, B, C, D, E, and G).
Fig 2: PEDF inhibits the activation of the NLRP3 inflammasome and reduces downstream increases in IL-18 and IL-1β through downregulation of CMPK2 in the HTR8/SVneo human chorionic trophoblast cell line. (A) The NLRP3 inflammasome in HTR8/SVneo cells was immunoprecipitated using an NLRP3 antibody, and the cell lysate input controls were tested by western blotting. The HTR8/SVneo cells were treated with 10 μg/ml LPS for 6 h. Densitometry analysis of (B) PEDF and CMPK2, (C) NLRP3, ASC and Casp1, and (D) pro-IL-18 and pro-IL-1β protein expression in HTR8/SVneo cells; n=5. (E) Relative mRNA levels of pro-IL-18 and pre-IL-1β in HTR8/SVneo cells; n=5. (F) The protein concentration of IL-18 and IL-1β in the cultured supernatants was analyzed by ELISA. n=5. **P<0.01 vs. relative normal group; #P<0.05, ##P<0.01 vs. relative LPS group; &P<0.05, &&P<0.01 vs. relative LPS + PEDF group. PEDF, pigment epithelium-derived factor; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; LPS, lipopolysaccharide; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CMPK2, cytidine monophosphate kinase 2; Casp1, caspase 1; CMPK2 group, LPS + PEDF-LVs + CMPK2-LV; LV, lentivirus; IP, immunoprecipitation; IB, immunoblotting.
Fig 3: PEDF alleviates LPS-induced mitochondrial and cellular damage by decreasing the expression of CMPK2 in HTR8/SVneo cells. (A) Fluorescence microscopy for Mito-SOX Red-labelled ROS in cytosol of HTR8/SVneo cells. Scale bar, 100 μm. HTR8/SVneo cells, cells overexpressing PEDF, or cells overexpressing CMPK2, were treated with 10 μg/ml LPS for 6 h. (B) Quantification of fluorescence density of mtROS in HTR8/SVneo cells. n=5. (C) Cytosolic mtDNA copy numbers were measured using by quantitative PCR; n=5. HTR8/SVneo (D) cell death and (E) cell viability were determined by assessing LDH release or using a Cell Counting Kit-8 assay, respectively. n=5. **P<0.01 vs. relative normal group; ##P<0.01 vs. relative LPS group; &&P<0.01 vs. relative LPS + PEDF group. PEDF, pigment epithelium-derived factor; LPS, lipopolysaccharide; CMPK2, cytidine monophosphate kinase 2; mt, mitochondrial; ROS, reactive oxygen species; LDH, lactate dehydrogenase.
Fig 4: Effects of CMPK2 KO on DENV infection-induced mitochondrial superoxide production and inflammasome pathway(A−G) THP-1 cells and THP-1 CMPK2-KO clones were infected with DENV (MOI = 5), and the levels of mtROS production measured by staining with MitoSOX were analyzed with flow cytometry (A). The production of mtROS in BMDCs transfected with control siRNA or CMPK2-specific siRNA and then infected with DENV (MOI = 1) was determined by flow cytometry (B). The effect of CMPK2 knockdown on DENV-induced mtROS production in human DCs was also determined (C). THP-1 cells and THP-1 CMPK2-KO clones were infected with DENV, and the levels of mature IL-1β in the supernatant were measured by western blotting (D) and ELISA (E). Additionally, the protein and mRNA expression of IL-1β in BMDCs transfected with control siRNA or CMPK2-specific siRNA and then infected by DENV was determined by western blotting (F). The effect of CMPK2 knockdown on DENV-induced IL-1β protein production in human DCs was examined with ELISA (G). Values represent the mean of the individual measurements in each sample ±SEM. ∗p < 0.05 and ∗∗p < 0.01. p value was calculated by the Student's t test (A, B, C, E, and G).
Fig 5: Activation of the NLRP3 inflammasome was observed in the decidua and chorion tissues of MA patients. (A) Immunohistochemistry staining of NLRP3 in the decidua and chorion tissues from patients who underwent IA or MA. Scale bar, 100 μm. (B) Ratio of moderate and strong expression to negative and weak expression of NLRP3 in decidua and chorion tissues; n=20. (C) Western blot of an IP of the NLRP3 inflammasome in the decidua and chorion tissues and the cell lysate input controls. Quantitative analysis of (D) PEDF and CMPK2, and (E) NLRP3, ASC and Casp1 protein expression levels; n=20. *P<0.05, **P<0.01 vs. indicated or relative IA group; #P<0.05 vs. relative decidua tissue of MA patients. MA, missed abortion; IA, induced abortion; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3; IP, immunoprecipitation; IB, immunoblotting; PEDF, pigment epithelium-derived factor; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; CMPK2, cytidine monophosphate kinase 2; Casp1, caspase1.
Supplier Page from Abcam for Anti-CMPK2 antibody