Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: DENV infection induced CMPK2 (A−F) Human dendritic cells (DCs) were infected with DENV (MOI = 5) or mock infected for 24 h, and microarray analysis was conducted. Among the 20 listed mitochondria-associated genes, CMPK2 was the one most highly induced by DENV infection (A). Both human and mouse primary and immortalized cells, including human DCs, BMDCs, BMDMs, and the cell lines A549, 293T, and THP-1, were infected with DENV at an MOI of 1 (A549 and 293T), MOI of 5 (human DCs, BMDCs, and BMDMs), or different MOIs (THP-1 cells). THP-1 cells were infected for 72 h, and the other cells were infected for 24 h. The mRNA expression of CMPK2 was measured by qPCR (B). The protein levels of CMPK2 in THP-1 cells, BMDCs, and A549 cells infected with various MOIs of DENV or treated with IFN-α (100 U/mL) as indicated were determined by western blotting (C) and confocal microscopy with staining with an anti-CMPK2 antibody, MitoTracker Deep Red, anti-NS3, and DAPI (D). Scale bar, 5 μm. The CMPK2 protein was detectable in both cytosolic and mitochondrial subfractions of A549 cells infected by DENV (MOI = 1) (E). BMDCs infected with four different DENV serotypes were evaluated to measure the mRNA expression of CMPK2 (F). Values represent the mean of the individual measurements in each sample ±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. p value was calculated by the Student's t test (B, C, E, and F), except for THP-1 cells in (B) where one-way ANOVA was used for calculation.

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Infection, Microarray, Expressing, Western Blot, Confocal Microscopy, Staining

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: Impacts of CMPK2 knockdown or overexpression on DENV infection-induced effects (A−G) BMDMs, BMDCs, or human DCs were treated with small interfering RNA (siRNA) targeting CMPK2 to KD the expression of CMPK2 or control siRNA. Cells were then infected with DENV (MOI = 0.1 for murine cells and MOI = 5 for human DCs) for 48 h (murine cells) or 24 h (human DCs), and viral RNA and CMPK2 mRNA levels were measured (A for BMDMs, B for BMDCs, and C for human DCs). A549 cells were transfected with CMPK2-GFP or a GFP control and then infected with DENV, and the expression of CMPK2-GFP and viral NS2B was measured by western blotting (D). A549 cells were transfected with different doses of CMPK2-GFP or the GFP control and then infected with DENV (MOI = 1) or left uninfected, and the expression of CMPK2-GFP and viral NS2B was measured by western blotting (E). A549 cells with CMPK2 KD by treatment with siRNA were infected with DENV (MOI = 1) in the presence or absence of different dosages of IFN-α as indicated. The expression of CMPK2 and viral NS2B was measured by western blotting (F). BMDCs infected with DENV for 48 h were collected, and cell survival was measured with a CCK-8 assay (G). Values represent the mean of the individual measurements in each sample ±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. p value was calculated by the Student's t test (A, B, C, D, and G) or one-way ANOVA (E).

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Knockdown, Over Expression, Infection, Small Interfering RNA, Expressing, Control, Transfection, Western Blot, CCK-8 Assay

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: Effects of CMPK2 knockout in the context of DENV infection (A−G) Four THP-1 CMPK2-KO clones were generated with CRISPR-Cas9 approaches as described in experimental procedures (A). DENV infection (MOI = 5) failed to induce the protein expression of CMPK2 in all CMPK2-KO clones; IFN-α treatment served as a positive control (B). The DENV infection-induced mRNA and protein expressions of both IFN-α and IFN-λ1 were evaluated in wild-type cells and CMPK2-KO clones (C). The mRNA levels of IFN-λ2/3, IFN-α, and tumor necrosis factor alpha (TNF-α) were measured in BMDCs with or without CMPK2 KD infected with DENV (MOI = 1) for 24 h (D). Human DCs treated with siCtl or siCMPK2 were infected by DENV (MOI = 5) for 24 h, and the supernatants were collected for measuring IFN-α, IFN-λ1, and TNF-α protein concentrations with ELISA (E). THP-1 and four THP-1 CMPK2-KO clones (F) and BMDCs (G) were infected by DENV (MOI = 5 for THP-1 and MOI = 1 for BMDCs), and the expression of CMPK2, un-phosphorylated, and phosphorylated p65 were measured by western blots. Values represent the mean of the individual measurements in each sample ±SEM. The representative results from two independent experiments were shown (F and G). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. p value was calculated by the Student's t test (C, D, and E).

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Knock-Out, Infection, Clone Assay, Generated, CRISPR, Expressing, Positive Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: 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).

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Infection, Activation Assay, Clone Assay, Control, Expressing, Flow Cytometry, Knockdown, Confocal Microscopy

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: 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).

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Infection, Clone Assay, Staining, Flow Cytometry, Transfection, Control, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: The effects of IFN-α receptor or STAT1 KO on DENV-induced CMPK2 expression and related events (A−E) BMDCs were prepared from mice with KO of IFN-αR or STAT1 or control mice. Cells were then infected with DENV (MOI = 1), and the expression of CMPK2, NS2B, phosphorylated STAT1, total STAT1, IRF1, and β-actin was measured by western blotting (A). The expression of CMPK2 mRNA and DENV RNA was determined by qPCR (B). In addition, the production of mtROS (C) and 8-OHdG (D) was measured. Similar to Figure 4 , the effects of IFN-αR or STAT1 KO on DENV-induced mtDNA release into the cytosol were determined in BMDCs (E). 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 (B, C, D, and E).

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Expressing, Control, Infection, Western Blot

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet: Overexpression of CMPK2 partially restored the antiviral effect of BMDCs from IFN-αR-KO mice (A−D) BMDCs prepared from mice with IFN-αR knocked out or control mice were transfected with a lentivirus carrying DYK or CMPK2-DYK, and the cells were then infected with DENV (MOI = 1). The expression of DENV RNA, CMPK2 mRNA, IFN-α mRNA, and IFN-λ2/λ3 mRNA was determined by qPCR (A). Additionally, the levels of DENV NS1 protein expression were determined by flow cytometry, and the percentages of NS1-positive cells and the relative mean fluorescence intensity (MFI) of NS1 are presented individually (B). (C) The transduction efficiency from six independent experiments is shown. The percentages of CD11c-positive cells after transfection with DYK or CMPK2-DYK were measured by flow cytometry (D). Analysis of six independent experiments was performed. Values represent the mean of the individual measurements in each sample ±SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. p value was calculated by the Student's t test (A, B, C, and D).

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Over Expression, Control, Transfection, Infection, Expressing, Flow Cytometry, Fluorescence, Transduction

Journal: iScience

Article Title: Mitochondrial CMPK2 mediates immunomodulatory and antiviral activities through IFN-dependent and IFN-independent pathways

doi: 10.1016/j.isci.2021.102498

Figure Lengend Snippet:

Article Snippet: CMPK2 (Confocal Microscopy) , GeneTex , Cat# GTX31502.

Techniques: Western Blot, Confocal Microscopy, Purification, Control, Virus, Recombinant, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software