Journal: Biology

Article Title: The Metabolic Activation of Sofosbuvir Is Impaired in an Experimental Model of NAFLD

doi: 10.3390/biology11050693

Figure Lengend Snippet: Sequences of the Primers Used in the Study for qRT-PCR Experiments.

Article Snippet: After a blocking step performed with 10% skim milk in TBS-T, the membrane was incubated overnight at 4 degrees with a primary anti-UMP-CMPK1 antibody (Santa Cruz Biotechnology; Dallas, TX, USA, diluted 1:500), and then with a secondary anti-mouse HRP-conjugated antibody (LGC Seracare; Milford, MA, USA, diluted 1:100).

Techniques:

Journal: Biology

Article Title: The Metabolic Activation of Sofosbuvir Is Impaired in an Experimental Model of NAFLD

doi: 10.3390/biology11050693

Figure Lengend Snippet: mRNA expression of the enzymes UMP-CMPK1 ( A ) and NDPK ( B ), responsible for the activation of sofosbuvir to the active compound GS-331007-TP. Protein expression of the enzyme UMP-CMPK1 ( C ). A representative Western Blot is shown in ( D ). Results are reported as means and S.E.M. of 6 animals per group. * p < 0.05 vs. healthy rats.

Article Snippet: After a blocking step performed with 10% skim milk in TBS-T, the membrane was incubated overnight at 4 degrees with a primary anti-UMP-CMPK1 antibody (Santa Cruz Biotechnology; Dallas, TX, USA, diluted 1:500), and then with a secondary anti-mouse HRP-conjugated antibody (LGC Seracare; Milford, MA, USA, diluted 1:100).

Techniques: Expressing, Activation Assay, Western Blot

Journal: ACS pharmacology & translational science

Article Title: All Blood Brain Barrier Cell Types Demonstrate Capability to Influence Differential Tenofovir and Emtricitabine Metabolism and Transport in the Brain.

doi: 10.1021/acsptsci.4c00510

Figure Lengend Snippet: Figure 2. BBB cells differentially express TFV and FTC transporters and nucleotide-metabolizing kinases. Primary human BBB cell monoculture and hepatocyte lysates were processed for LC−MS/MS-based proteomics and protein concentrations for (A) ENT1, (B) MRP4, and (C) MRP1 TFV/FTC transporters, (D) AK2, (E) CKB, (F) PKLR, and (G) PKM TFV nucleotide-metabolizing kinases, as well as (H) CMPK1, (I) DCK, (J) PGK1, and (K) TK1 FTC nucleotide-metabolizing kinases, each with respective protein Western blots to confirm protein abundance in BBB cells. Concentrations of proteins of interest were measured by Proteomic Ruler. Four to five independent experiments with two LC−MS/MS injection replicates each were performed (represented as individual dots). Replicates with missing LC−MS/MS values were omitted from the plot. >2 independent experiments with missing values were reported as not reliably detected (ND) by proteomics analyses. Data represented as mean ± standard deviation. Statistical analysis was performed using Brown−Forsythe and Welch ANOVA or Kruskal−Wallis ANOVA by GraphPad software. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Western blots were performed as previously described.16 Blots were probed with antibodies with specificity to BCRP (NBP2-22124, Novus Bio, Centennial, CO), P-gp (PA5-61300, Invitrogen), MRP4 (12705, Cell Signaling Technology), MRP1 (Ab24102, Abcam), ENT1 (PA5-116451, Invitrogen), PGK1 (PA528612, Invitrogen), AK2 (11014-1-AP, Proteintech, Rosemont, IL), CKB/CKM (15137-1-AP, Proteintech), TK (15691-1-AP, Proteintech), CMPK (11360-1-AP, Proteintech), DCK (17758-1-AP, Proteintech), PKM1 (15821-1-AP, Proteintech), and PKLR (2456-1-AP, Proteintech), overnight at 4 °C, washed with TBS-T, and probed with the appropriate secondary antibody (ab97023 and Ab97051, Abcam) for 1 h at room temperature.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Quantitative Proteomics, Injection, Standard Deviation, Software

Journal: ACS pharmacology & translational science

Article Title: All Blood Brain Barrier Cell Types Demonstrate Capability to Influence Differential Tenofovir and Emtricitabine Metabolism and Transport in the Brain.

doi: 10.1021/acsptsci.4c00510

Figure Lengend Snippet: Figure 4. BBB TFV/FTC nucleotide-metabolizing kinases and transporters are impacted in a cell-dependent manner by ART and HIV. Primary human pericyte and astrocyte monocultures were exposed to ART (10 μM TFV, 10 μM FTC, and 10 μM DTG), HIV (5 ng/mL), or HIV + ART (10 μM TFV, 10 μM FTC, 10 μM DTG, and 5 ng/mL HIV) for 24 h at 37 °C, 5% CO2. Treatment with a vehicle was used as a control. Pericytes and astrocytes were lysed and processed for proteomics analyses. Concentrations of proteins of interest were measured by Proteomic Ruler. After exposure, changes in protein concentration were assessed in (A−G) astrocytes (astro) for (A) AK2, (B) CMPK1, (C) ENT1, (D) P-gp, (E) PGK1, (F) MRP4, and (G) MRP1. Similarly, changes in protein concentration were assessed in (H−L) pericytes (per) for (H) CKB, (I) MRP4, (J) MRP1, (K) PGK1, and (L) TK1. The significant changes (A−L) in the concentrations of TFV and FTC transporters and metabolizing enzymes after ART, HIV, or HIV + ART exposure relative to vehicle were (M) summarized for astrocytes (astro) and pericytes (per). An arrow next to each protein denotes an increase (upward arrow) or decrease (downward arrow) in protein concentration after ART, HIV, or HIV + ART exposure relative to the vehicle. Four independent experiments with two LC−MS/MS injection replicates each were performed per condition (represented as individual dots). Replicates with missing LC−MS/MS values were omitted from the plot. >2 independent experiments with missing values were reported as not reliably detected (ND) by proteomics analyses. Data represented as mean ± standard deviation. Statistical analysis was performed using Brown−Forsythe and Welch ANOVA or Kruskal−Wallis ANOVA by GraphPad software. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Western blots were performed as previously described.16 Blots were probed with antibodies with specificity to BCRP (NBP2-22124, Novus Bio, Centennial, CO), P-gp (PA5-61300, Invitrogen), MRP4 (12705, Cell Signaling Technology), MRP1 (Ab24102, Abcam), ENT1 (PA5-116451, Invitrogen), PGK1 (PA528612, Invitrogen), AK2 (11014-1-AP, Proteintech, Rosemont, IL), CKB/CKM (15137-1-AP, Proteintech), TK (15691-1-AP, Proteintech), CMPK (11360-1-AP, Proteintech), DCK (17758-1-AP, Proteintech), PKM1 (15821-1-AP, Proteintech), and PKLR (2456-1-AP, Proteintech), overnight at 4 °C, washed with TBS-T, and probed with the appropriate secondary antibody (ab97023 and Ab97051, Abcam) for 1 h at room temperature.

Techniques: Control, Protein Concentration, Liquid Chromatography with Mass Spectroscopy, Injection, Standard Deviation, Software

Journal: Frontiers in Oncology

Article Title: CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer

doi: 10.3389/fonc.2021.637470

Figure Lengend Snippet: Identification of the CMPK1 affect the 5-FU chemosensitivity. (A) Kaplan-Meier survival curves of progression-free survival (PFS) for 68 GC patients with 5-FU-based-chemotherapy response (yellow line) or non-response (blue line). (B) Association of clinical features (age, gender, survival status, molecular subtype) and treatment response in GC samples. (C) Dysregulation of signaling pathways stratified by response versus non-response subtypes. (D) volcano plot of the differentially expressed genes in different treatment response subtypes. Blue and red dot indicated the genes highly expressed in non-response and response subgroups, respectively. (E) Kaplan-Meier survival analysis of PFS based on CMPK1 expression subgroups. The samples with upper two thirds CMPK1 expression were termed as high expression, the remaining were termed as low expression. (F) Distribution of TYMS in CMPK1 high versus low expression subgroup (G) Top enriched gene pathways in distinct CMPK1 expression subgroups (high vs low) were assessed by using the GSEA algorithm.

Article Snippet: The transfection of miR-130b mimic with miRNA control (miR-NC) (Ambion, USA) and CMPK1 siRNA with control siRNA (Shanghai GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine RNAiMAX (Invitrogen, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer

doi: 10.3389/fonc.2021.637470

Figure Lengend Snippet: CMPK1 affects gastric cancer cell line sensitivity to 5-FU treatment. (A, B) AGS and MGC803 cells were transfected with 20 nM si-NC or si-CMPK1. After 48 h, cells were harvested for western-blot analysis (A) or RT-qPCR experiments (B) . (C) Cell viability of si-NC and si-CMPK1 with5-FU treatment was tested by MTT assay. (D) 5-FU sensitivity detection was re-confirmed in clonogenic cell-survival assay. (E, F) Apoptosis and cell-cycle analysis were pre-treated as previously described and measured by flow cytometry to determine the impact of CMPK1 treated with or without 5-FU. The representative flow cytometry patterns of cell cycle distribution and the statistical analysis was shown in (E) . The representative flow cytometry patterns of cell apoptosis and the statistical analysis was shown in (F) . (G) Knock-down of CMPK1 suppressed 5-FU-induced DNA damage determined by comet assay in AGS cells. Data are presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Article Snippet: The transfection of miR-130b mimic with miRNA control (miR-NC) (Ambion, USA) and CMPK1 siRNA with control siRNA (Shanghai GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine RNAiMAX (Invitrogen, USA).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, MTT Assay, Clonogenic Cell Survival Assay, Cell Cycle Assay, Flow Cytometry, Single Cell Gel Electrophoresis

Journal: Frontiers in Oncology

Article Title: CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer

doi: 10.3389/fonc.2021.637470

Figure Lengend Snippet: Prediction and validation of CMPK1 upstream regulator miR-130b in gastric cancer. (A) A Venn diagram showing the combination of four miRNA prediction algorithms identified three candidate upstream regulators of CMPK1. (B–D) Inverse correlation between CMPK1 and miR-130b, miR-519, and miR-17 in GC tissue (E) RT-qPCR analysis revealed that transfection with miR-130b mimic decreased the CMPK1 RNA level. (F) The protein level of CMPK1 was decreased in MGC-803 and AGS cells when transfected with miR-130b with β-actin as a loading control. (G) On the contrary, cells transfected with anti-miR-130b expressed increased levels of CMPK1 protein compared to those transfected with anti-miR-NC. (H) A putative miR-130b -binding site exists in the 3′-UTR of the CMPK1 mRNA, and 7-nucleotide deletion were generated in the binding site. (I) MGC-803 and (J) AGS cells transfected with pmirGLO-CMPK1-Wild and pmirGLO-CMPK1-Mut reporters, together with a miR-130b mimic or negative control, miR-130b overexpression suppressed the activity of luciferase in the wild-type but not in mutant type. All the values shown were represented as means ± SD. (*P < 0.05; **P < 0.01 vs negative control by two-tailed Student’s t-test).

Article Snippet: The transfection of miR-130b mimic with miRNA control (miR-NC) (Ambion, USA) and CMPK1 siRNA with control siRNA (Shanghai GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine RNAiMAX (Invitrogen, USA).

Techniques: Quantitative RT-PCR, Transfection, Binding Assay, Generated, Negative Control, Over Expression, Activity Assay, Luciferase, Mutagenesis, Two Tailed Test

Journal: Frontiers in Oncology

Article Title: CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer

doi: 10.3389/fonc.2021.637470

Figure Lengend Snippet: MiR-130b has an influence on 5-FU sensitivity. (A, B) AGS and MGC-803 cells were transfected with either miR-NC or miR-130b mimic for 48h, and cells were reseeded for 5-FU sensitivity detection using an MTT assay (A) and a clonogenic cell-survival assay (B) . (C) AGS and MGC-803 cells were transfected with either anti-miR-NC or anti-miR-130b upon 5-FU treated. Cell viability was assessed by MTT assay. (D–F) Cells in the apoptosis and cell-cycle analysis were pre-treated and measured by flow cytometry. AGS cells were used to determine the impact of miR-130b/control treated with 5-FU. The representative flow cytometry patterns of cell cycle distribution is shown in (D) and the statistical analysis is shown in (E) . The representative patterns of cell apoptosis are shown in ( F , upper panel) and the statistical analysis is shown in ( F , lower panel). (G) Overexpression of miR-130b in AGS cells and 5-FU caused DNA damage detected by comet assay. Representative images are shown in ( G , left panel) and the mean ± SD for each condition shown in ( G , right panel). (H, I) AGS cells were cotransfected with the CMPK1 CDS region or negative control vector together with 20 nM miR-NC or miR-130b. After 24 h, cells were harvested for western blot analysis (H) or reseeded for 5-FU sensitivity assay (I) . Data represent the mean ± SD, *P < 0.05; **P < 0.01, two-sided Student’s t test.

Article Snippet: The transfection of miR-130b mimic with miRNA control (miR-NC) (Ambion, USA) and CMPK1 siRNA with control siRNA (Shanghai GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine RNAiMAX (Invitrogen, USA).

Techniques: Transfection, MTT Assay, Clonogenic Cell Survival Assay, Cell Cycle Assay, Flow Cytometry, Over Expression, Single Cell Gel Electrophoresis, Negative Control, Plasmid Preparation, Western Blot, Sensitive Assay

Journal: Frontiers in Oncology

Article Title: CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer

doi: 10.3389/fonc.2021.637470

Figure Lengend Snippet: MiR-130b-mediated regulation of CMPK1 influences chemoresistance to 5-FU (A, B) AGS and MGC-803 cells were transfected with miR-NC, miR-130b mimic or si-CMPK1. After 48 h, cells were reseeded for cisplatin sensitivity detection by MTT assay. (C, D) The 3-D culture assay showed that 5-FU treatment, but not cisplatin, significantly impacted miR-130b/miR-NC/si-CMPK1 cell growth on martrigel matrix. AGS cell spheroids, morphological changes, and the diameter of clone is shown in (C, D) , MGC-803 cell spheroids are shown in (E, F) . (G) Representative images of orthotopic gastric cancer mouse model for 5-FU sensitivity in miR-NC, miR-130b, and CMPK1-treatment. The scale was millimeters. (H) Mean tumor volumes in mice treated with different combination of miR-NC, miR-130b, CMPK1 and 5-FU regimens. Expression level of miR-130b (I) and CMPK1 (J) in different xenograft mouse model by q-PCR analysis. (K) Tumor samples from control- and miR-130b-treated mice were sectioned and stained for CMPK1, TUNEL and Cleaved Caspase-3 by immunohistochemistry (IHC). (L) Quantification of CMPK1, TUNEL and Cleaved Caspase-3 positive cell level. Error bars, ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The transfection of miR-130b mimic with miRNA control (miR-NC) (Ambion, USA) and CMPK1 siRNA with control siRNA (Shanghai GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine RNAiMAX (Invitrogen, USA).

Techniques: Transfection, MTT Assay, Expressing, Staining, TUNEL Assay, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: CMPK1 Regulated by miR-130b Attenuates Response to 5-FU Treatment in Gastric Cancer

doi: 10.3389/fonc.2021.637470

Figure Lengend Snippet: Schematic of the proposed molecular mechanism of miR-130b in GC affecting 5-FU metabolism. First, 5-FU is converted to FUMP in vivo by OPRT. MiR-130b directly targets CMPK1 expression and suppresses the phosphorylation of 5-FU FUMP to the diphosphate metabolites FUDP and FdUDP. As a result, miR-130b attenuates the response of gastric cancer cells to chemotherapy and impacts survival. FUMP, 5-fluorouridine-5′-monophosphate; FUDP, 5-fluorouridine-5′-diphosphate; FdUDP, 5-fluoro-2′-deoxyuridine-5′-diphosphate.

Article Snippet: The transfection of miR-130b mimic with miRNA control (miR-NC) (Ambion, USA) and CMPK1 siRNA with control siRNA (Shanghai GenePharma, China) were performed according to the manufacturer’s instruction using Lipofectamine RNAiMAX (Invitrogen, USA).

Techniques: In Vivo, Expressing