Journal: Frontiers in Cell and Developmental Biology

Article Title: 3-Deoxysappanchalcone Inhibits Skin Cancer Proliferation by Regulating T-Lymphokine-Activated Killer Cell-Originated Protein Kinase in vitro and in vivo

doi: 10.3389/fcell.2021.638174

Figure Lengend Snippet: 3-Deoxysappanchalcone (3-DSC) inhibits T-LAK cell-originated protein kinase (TOPK) kinase activity by binding to Thr42 and Asn172. (A) The protein expression levels of TOPK and its downstream protein effectors in Normal Human Dermal Fibroblast (NHDF) and skin cancer cell lines (A431, SK-MEL2, SK-MEL-28, and A375) and colon cancer cell line HCT15 (positive control). (B,C) The expression levels of TOPK dependent on the tumor type (primary melanoma patients, metastatic melanoma patient, and normal person) or gender from The Cancer Genome Atlas-Skin Cutaneous Melanoma (TCGA-SKCM) cohort database. TOPK transcript expression was analyzed according to tissue type according to transcript per million (TPM) units. (D) Effect of 3-DSC on TOPK phosphorylation. Kinase assay was performed with active TOPK (100 ng), 3-DSC (2.5, 5, 10, or 20 μM), HI-TOPK-032 (5 μM), myelin basic protein (MBP) (400 ng), and ATP (40 μM). MBP is the substrate of TOPK kinase; p-Thr/Ser levels are an indicator of TOPK activity. The band intensity of p-Thr/Ser is significantly reduced when the assay was performed with inclusion of 2.5 μM 3-DSC. (E) Band quantification results of (D) . (F) Kinetic analysis of 3-DSC whether competing ATP with TOPK substrate. Km and Vm values of TOPK enzyme incubated with or without 3-DSC. (G) Binding affinity of 3-DSC to TOPK was assessed in four skin cancer cells. 3-DSC-Sepharose 4B or Sepharose 4B were incubated with cell lysates. Protein was then eluted from the beads and separated by SDS-PAGE. (H) Diagram illustrating the mutation of residues previously predicted to be important in facilitating the interaction between TOPK and 3-DSC. TOPK was mutated at the T42A, N172A, and T42A residues based upon computer docking analysis. (I) In vitro pull-down assay with TOPK WT, TOPK T42A, TOPK N172A, and TOPK T42A + N172A. Double mutation significantly attenuated binding of 3-DSC with TOPK. * p < 0.05, *** p < 0.001.

Article Snippet: Active TOPK kinase (100 ng, Cat#T14-10G), kinase dilution buffer (Cat#K01-09-20), and 10 mM ATP (Cat#A50-09) were purchased from SignalChem Biotech Inc. (Richmond, BC, Canada).

Techniques: Activity Assay, Binding Assay, Expressing, Positive Control, Phospho-proteomics, Kinase Assay, Incubation, SDS Page, Mutagenesis, In Vitro, Pull Down Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

doi: 10.1016/j.omtn.2022.01.004

Figure Lengend Snippet: Overview of in vitro transcription process and chemical modifications of IVT-mRNA used in this study Main steps, and conditional post-transcriptional treatments, which were only applied to samples with cap modifications, i.e., methylation and dephosphorylation, are illustrated at the top panel. Simplified chemistry of IVT-mRNA synthesized and utilized at this study with various cap and/or nucleotide modifications are depicted at the bottom panel. Sequence of synthesis steps applied for cap modified mRNAs are indicated as colored bars corresponding to each synthesis step. Phosphatase treatment step is further indicated with/− or/+ next to sample name. Chemical formula only indicate the main variations among nucleotides as well as cap structures. See Figure S1 for precise chemical formula of cap and nucleotide modifications, and for detailed information on chemistry and synthesis process of each sample. pDNA, plasmid DNA; T7 Pol., T7 RNA polymerase; NTs, nucleotides; ARCA, anti-reverse cap analog; Ψ, pseudouridine; me 1 Ψ, N 1 -Methylpseudouridine; 5moU, 5-methoxy-uridine; 5meC, 5-methyl-cytidine.

Article Snippet: Hereby, uridine is fully substituted either by Ψ, N1-methyl-pseudouridine (me 1 Ψ) (Jena Bioscience) or 5-methoxy-uridine (5moU) (Jena Bioscience); cytidine is fully substituted by 5meC.

Techniques: In Vitro, Methylation, De-Phosphorylation Assay, Synthesized, Sequencing, Modification, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

doi: 10.1016/j.omtn.2022.01.004

Figure Lengend Snippet: IVT-mRNA double strand content and integrity investigated by dot blot and agarose gel electrophoresis (A) Poly(A) as ssRNA-negative control, and dsRNA positive control were blotted with the same amount as main samples (1,000 ng/dot), next to dsRNA gradient of 4-fold serial dilutions for generating a standard curve for subsequent quantifications and detected by a dsRNA-antibody. Representative dot blots of IVT-mRNAs with different (B) cap modifications, and (C) nucleotide modifications presented side-by-side with denatured agarose gel electrophoresis images of the same samples. Quantified dsRNA for IVT-mRNAs with various cap modifications (D) as well as (E) nucleotide modifications plotted as weight percent of dsRNA content (calculated according to positive control standard curve) to total mRNA amount blotted on membrane for each sample. Error bars indicate SEM for three independently synthesized IVT-mRNA batches blotted on the membrane in duplicates; see Figure S2 for uncropped membrane and the gel image. ARCA, anti-reverse cap analog; MT, methyl-transferase; AnP, Antarctic phosphatase; Ψ, pseudouridine; me 1 Ψ, N 1 -Methylpseudouridine; 5moU, 5-methoxy-uridine; 5meC, 5-methylcytidine.

Article Snippet: Hereby, uridine is fully substituted either by Ψ, N1-methyl-pseudouridine (me 1 Ψ) (Jena Bioscience) or 5-methoxy-uridine (5moU) (Jena Bioscience); cytidine is fully substituted by 5meC.

Techniques: Dot Blot, Agarose Gel Electrophoresis, Negative Control, Positive Control, Synthesized

Journal: Molecular Therapy. Nucleic Acids

Article Title: Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

doi: 10.1016/j.omtn.2022.01.004

Figure Lengend Snippet: Transfection efficiency and EGFP mRNA expression level in macrophages transfected with IVT-mRNA with various cap and nucleotide modifications (A) Representative fluorescent images and (B) flow cytometric density plots indicating EGFP expression in macrophages transfected with low dose and high dose of IVT-mRNA made of either Cap 0 (i.e., ARCA), or Cap 1 (i.e., ARCA + MT, and CleanCap) with and without phosphatase treatment. (C) Quantification of transfection efficiency, and (D) EGFP mRNA expression level in macrophages transfected with low doses and high doses of the different IVT-mRNAs with cap modifications. (E) Representative fluorescent images of macrophages transfected with low doses of IVT-mRNA composed of nucleotides with different chemical modifications. (F) Flow cytometric density plots indicating EGFP expression in macrophages transfected with low dose and high dose of IVT-mRNA with various nucleotide modifications. (G) Transfection efficiency and (H) EGFP mRNA expression level quantified by flow cytometry and plotted in terms of EGFP-positive cells percentage and MFI of EGFP signal among EGFP-positive cell populations, respectively. Poly(I:C) was also transfected in low dose (125 ng∙mL −1 ). For each condition 125 ng∙mL −1 and 500 ng∙mL −1 of IVT-mRNA were used for transfection referred here as low dose and high dose, respectively. Values are presented as mean ± SD. Error bars indicate SD of three independent experiments from three individual donors. Bar = 50 μm. Statistical differences are depicted with ∗∗p < 0.005, ∗∗∗p < 0.001. ARCA, anti-reverse cap analog; MT, methyl-transferase; AnP, Antarctic phosphatase; Ψ, pseudouridine; me 1 Ψ, N 1 -Methylpseudouridine; 5moU, 5-methoxy-uridine; 5meC, 5-methyl-cytidine.

Article Snippet: Hereby, uridine is fully substituted either by Ψ, N1-methyl-pseudouridine (me 1 Ψ) (Jena Bioscience) or 5-methoxy-uridine (5moU) (Jena Bioscience); cytidine is fully substituted by 5meC.

Techniques: Transfection, Expressing, Flow Cytometry

Journal: Molecular Therapy. Nucleic Acids

Article Title: Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

doi: 10.1016/j.omtn.2022.01.004

Figure Lengend Snippet: Evaluation of CD80 expression in macrophages, in response to IVT-mRNA transfection with different chemistry Staggered histogram of CD80 levels in macrophages transfected with low doses and high doses of IVT-mRNA with various cap modifications (A), as well as nucleotide modifications (C) along with untransfected, poly(I:C), the dsRNA positive control transfected in low dose, and activated macrophages. Activated cells were treated with LPS/IFN-γ. MFI of CD80 normalized to untransfected cells is indicated for macrophages transfected with low doses and high doses of mRNA with different cap modifications (B) and nucleotide modifications (D). Mock transfection refers to carrier (i.e., LipoMM) without mRNA. For each condition 125 ng∙mL −1 and 500 ng∙mL −1 of IVT-mRNA were used for transfection, referred here as low dose and high dose, respectively. Expression was measured 24 h after transfection. Values are presented as mean ± SD. Error bars indicate SD of three independent experiments from three individual donors (n = 3). ARCA, anti-reverse cap analog; MT, methyl-transferase; AnP, Antarctic phosphatase; Ψ, pseudouridine; me 1 Ψ, N 1 -Methylpseudouridine; 5moU, 5-methoxy-uridine; 5meC, 5-methyl-cytidine.

Article Snippet: Hereby, uridine is fully substituted either by Ψ, N1-methyl-pseudouridine (me 1 Ψ) (Jena Bioscience) or 5-methoxy-uridine (5moU) (Jena Bioscience); cytidine is fully substituted by 5meC.

Techniques: Expressing, Transfection, Positive Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages

doi: 10.1016/j.omtn.2022.01.004

Figure Lengend Snippet: Cytokine secretion in macrophages transfected with IVT-mRNA with various nucleotide modifications TNF-α secretion was measured (A) 6 h and (B) 24 h after transfection with low doses and high doses of different IVT-mRNA formula. IL-6 secretion was quantified (C) 6 h and (D) 24 h after transfection. IFN-β secretion was investigated (E) 6 h and (F) 24 h upon transfection. Mock transfection refers to carrier (i.e., LipoMM) without mRNA. Poly(I:C) was also transfected in low-dose positive control. For each condition, 125 ng∙mL −1 and 500 ng∙mL −1 of IVT-mRNA were used for transfection, referred here as low dose and high dose, respectively. Values are presented as mean ± SD. Error bars indicate SD of three independent experiments from three individual donors (n = 3). Ψ, pseudouridine; me 1 Ψ, N 1 -Methylpseudouridine; 5moU, 5-methoxy-uridine; 5meC, 5-methyl-cytidine.

Article Snippet: Hereby, uridine is fully substituted either by Ψ, N1-methyl-pseudouridine (me 1 Ψ) (Jena Bioscience) or 5-methoxy-uridine (5moU) (Jena Bioscience); cytidine is fully substituted by 5meC.

Techniques: Transfection, Positive Control