Journal: BMC pharmacology & toxicology

Article Title: Narciclasine induces autophagy-mediated apoptosis in gastric cancer cells through the Akt/mTOR signaling pathway.

doi: 10.1186/s40360-021-00537-3

Figure Lengend Snippet: Fig. 4 Narciclasine promotes autophagy of gastric cancer cells. A-C, Laser confocal scanning microscopy was used to observe the formation of autolysosomes in gastric cancer cells treated with narciclasine (0.5 μM) for 24 h. D-F, Western blotting was used to detect the effect of narciclasine combined with autophagy inhibitor 3-MA (5 mM) and CQ (2.5 μM) on LC3-II and p62 protein after treatment of gastric cancer cells for 24 h. Data are shown as mean ± SD. NCS: narciclasine; 3-MA: 3-methyladenine; CQ: chloroquine

Article Snippet: The human gastric cancer cell lines BGC-823, MGC-803, GES-1, MKN28 and SGC-7901 were purchased from the Institute of Biochemistry and Cell Biology at the Chinese Academy of Sciences (Shanghai, China); Roswell Park Memorial Institute 1640 (RPMI 1640), fetal bovine serum (FBS), penicillin and streptomycin from Gibco Life Technologies (NY, US); chloroquine (CQ) and 3-methyladenine (3-MA) from Selleck Chemicals (Texas, US); insulin from Sigma-Aldrich (CA, US); and AktiVIII from Macklin Inc. (Beijing, China).

Techniques: Confocal Laser Scanning Microscopy, Western Blot

Journal: Advanced Science

Article Title: Electrochemically Synthesis of Nickel Cobalt Sulfide for High‐Performance Flexible Asymmetric Supercapacitors

doi: 10.1002/advs.201700375

Figure Lengend Snippet: The XRD patterns a) of Ni–Co–S/GF and GF. Raman spectra b) obtained for PPy/GF, Ni–Co–S/GF, GF, and PPy (inset figure (b)). XPS spectrum of Ni–Co–S/GF: c) Ni 2p, d) Co 2p, e) S 2p, (f) C 1s and inset figure (f) is XPS survey spectrum of Ni–Co–S/GF.

Article Snippet: TEM (JEOL‐2100) and high‐resolution transmission electron microscopy (HRTEM; JEOL JEM‐2010F), Raman spectra were obtained via a confocal microscope Raman spectrometer system (Witec Alpha 300; with the wavelength excitation laser of 488 nm) and XRD (Bruker Focus D8 with Cu Kα radiation, Smart lab).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: In vitro susceptibilities of planktonic S. mutans UA159

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Comparative killing kinetics of CLP-4. S. mutans UA159 cultures at a cell density of 6 × 105 CFU/ml were challenged with 5, 10, and 25 μg/ml CLP-4 under conditions of active growth in CDM supplemented with 0.5% (wt/vol) glucose (A) and against growth-arrested cells in CDM lacking any carbon source (B). Samples at time zero were enumerated prior to peptide treatment. Data shown are the means and standard deviations of three biological replicates from three independent experiments.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: CLP-4 prevents S. mutans biofilm formation. (A) Biofilms inoculated with 2 × 107 CFU/ml were grown for 24 h in the presence of CLP-4, chlorhexidine, or erythromycin at concentrations ranging between 0.6× and 2× their respective MICs. Biofilm formation was quantified using crystal violet staining and expressed in percentage relative to untreated control. Shown are the means and standard deviations of three biological replicates from three independent experiments. *, P < 0.05; ***, P < 0.001 compared to untreated control. (B) Corresponding growth curve kinetics showing the MIC of CLP-4 on S. mutans UA159.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Effects of CLP-4 on preformed biofilms. S. mutans UA159 biofilms were established for 24 h and then treated with increasing concentrations (1× to 10× the MIC) of CLP-4, chlorhexidine, or erythromycin. (A) Antibiofilm activities were assessed by quantifying the cell viability of treated biofilms by colony enumeration on agar plates. The means and standard deviations of three biological replicates from three independent experiments are shown. **, P < 0.01; ***, P < 0.001 compared to untreated control. (B) Biofilms treated with 10× the MICs for each antimicrobial were fluorescently labeled using the LIVE/DEAD BacLight viability stain and visualized by confocal laser scanning microscopy. Shown are the top-down three-dimensional (3D) volume rendering of biofilms at a total magnification of ×400. Bottom images represent optical planes in the xz, and vertical thin images represent yz dimensions. Membrane-compromised bacteria are stained red with propidium iodide, while intact bacteria are stained green with SYTO 9. Areas highlighted by dashed lines indicate regions of interest (ROIs) viewed at a higher magnification. Dimensions shown are 387.5 μm by 387.5 μm by 16 μm. (C) ROIs are presented at ×2,300 magnification. Dimensions shown are 68.1 μm by 68.1 μm by 16 μm.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Control, Labeling, Staining, Confocal Laser Scanning Microscopy, Membrane, Bacteria

Journal: Journal of Cell Science

Article Title: Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4 + T cells through p65 and H2B phosphorylation

doi: 10.1242/jcs.181248

Figure Lengend Snippet: PKC-θ signaling and rapid transcriptional responses in memory CD4 + T cells. (A) A schematic of the in vitro transcriptional memory Jurkat T cell model: non-stimulated (NS) Jurkat T cells were activated with PMA and Ca 2+ ionophore (+P/I, denoted 1°) and then subjected to stimulus withdrawal (SW) for 9 days before re-stimulation (2°). (B) Venn diagram showing the number of genes grouped by their distinct transcriptional profiles in the Jurkat model. These profiles are for the primary-specific, activation-compliant, transcriptional-memory-responsive and secondary-specific groups. (C) Heatmap representation of inducible gene expression in naïve and memory CD4 + T cells treated with PKC-θ siRNA (siPKC) with and without PMA and Ca 2+ ionophore. Gene expression normalized to GAPDH is represented as z -scores (mean, n =2). The colors of the asterisk correspond to the gene groups shown in Fig. 1 B. siCtrl, control siRNA.

Article Snippet: Human naïve or memory CD4 + T cells were transfected for 48 h with PKC-θ (sc-36252, Santa Cruz Biotechnology) and FAM-labeled mock control siRNAs together or FAM-labeled negative control siRNA alone in Lipofectamine 2000 and Opti-MEM.

Techniques: In Vitro, Activation Assay, Gene Expression, Control

Journal: Journal of Cell Science

Article Title: Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4 + T cells through p65 and H2B phosphorylation

doi: 10.1242/jcs.181248

Figure Lengend Snippet: Identification of phosphorylated residues on histone H2B . (A) PKC-θ-mediated phosphorylation signals on H2B:21 (residues 21–40 derived from H2B) were detected by PKC-θ microarray profiling. The mean phosphorylation is shown (±s.d.). * denotes a pre-phosphorylated serine and the dotted red line is the background threshold (57500). The location of the histone H2B repression domain (HBR) is marked. (B) PKC-θ phosphorylates H2B Ser32. An in vitro kinase assay was performed by incubating active PKC-θ with either recombinant histones H2B, H3 or H4 or recombinant nucleosomes containing H3.1 or H3.3. Phosphorylated proteins were resolved by SDS-PAGE followed by western blotting for H2B phosphorylation. Negative controls include C1 (no ATP addition), C2 (no PKC addition), and C3 (incubation with PKC-μ). A representative blot of three experiments is shown. (C) PKC-θ phosphorylates H2B Ser36, as assessed by the method shown in B. (D) The Pearson's colocalization coefficient (PCC) was calculated for the fluorescent signal of H2B and PKC-θ as measured by confocal laser scanning microscopy in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations (mean±s.e.m., n =20). ** P ≤0.01, **** P ≤0.0001 (Mann–Whitney test). (E) Representative immunoblot of phosphorylated H2B Ser32 and Ser36 in DMSO and 1 μM C27-treated Jurkat T cells with or without PMA and Ca 2+ ionophore (PI) ( n =3). (F) Normalized H2B Ser32p densitometry is shown for human CD4 + T cells treated with either the control siRNA (siCtrl) or PKC-θ siRNA (siPKC-θ) (mean±s.e.m., n =3 individuals). *** P ≤0.001 (two-tailed Student's t -test). (G) FAIRE chromatin accessibility shown for IL2 and TNF in in non-stimulated (NS) Jurkat T cells, and cells after primary (1°) and secondary (2°) stimulations transfected with vector only (VO), wild-type PKC-θ plasmid (WT) or cytoplasmic-restricted PKC-θ mutant (NLS) plasmids. FAIRE chromatin accessibility is normalized to results for GAPDH (mean±s.e.m., n =3). *** P ≤0.001 (two-way ANOVA). (H) FAIRE chromatin accessibility shown for IL2 and other promoters in naïve and memory CD4 + T cells treated with either the control (siCtrl) or the PKC-θ siRNA (siPKC-θ) with or without PMA and Ca 2+ ionophore (P/I). FAIRE chromatin accessibility is normalized to GAPDH and expressed as a percentage relative to the stimulated (ST) memory CD4 + T cells treated with the control siRNA (siCtrl) (mean±s.e.m., n =3). * P ≤0.05, ** P <0.01 (unpaired two-tailed Student's t -test).

Article Snippet: Human naïve or memory CD4 + T cells were transfected for 48 h with PKC-θ (sc-36252, Santa Cruz Biotechnology) and FAM-labeled mock control siRNAs together or FAM-labeled negative control siRNA alone in Lipofectamine 2000 and Opti-MEM.

Techniques: Phospho-proteomics, Derivative Assay, Microarray, In Vitro, Kinase Assay, Recombinant, SDS Page, Western Blot, Incubation, Confocal Laser Scanning Microscopy, MANN-WHITNEY, Control, Two Tailed Test, Transfection, Plasmid Preparation, Mutagenesis

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