Journal: Journal of Gastrointestinal Oncology

Article Title: Expression and clinical significance of miR-17-5p in tumor tissues of patients with colorectal cancer

doi: 10.21037/jgo-22-1185

Figure Lengend Snippet: The effects of miR-17-5p on the invasive ability of HCT-116 cells. (A) Microscopic observation and photography after crystalline violet staining (100× magnification); (B) the OD values of each group at 570 nm wavelength after dissolution of crystalline violet by sodium acetate; (C-G) the relative mRNA and protein expression of miR-17-5p in HCT-116 cells was detected with RT-qPCR and western blot, respectively. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; in-NC, inhibitor negative control; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction; OD, optical density.

Article Snippet: The miR-17-5p overexpression mimics, inhibitor, and corresponding controls, hairpin miRNAs quantification kits, and U6 calibration real-time fluorescence quantitative PCR (RT-qPCR) kits were purchased from Genepharma Co. (Shanghai, China).

Techniques: Staining, Dissolution, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Journal of Gastrointestinal Oncology

Article Title: Expression and clinical significance of miR-17-5p in tumor tissues of patients with colorectal cancer

doi: 10.21037/jgo-22-1185

Figure Lengend Snippet: The effects of miR-17-5p on the apoptosis of HCT-116 cells. (A,B) Flow cytometric detection of the effects of different transfection groups on apoptosis of HCT-116 cells; (C-I) the relative mRNA and protein expression of miR-17-5p in HCT-116 cells was detected with RT-qPCR and Western blot, respectively. *, P<0.05; **, P<0.01; ***, P<0.001. NC, negative control; in-NC, inhibitor negative control; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction.

Article Snippet: The miR-17-5p overexpression mimics, inhibitor, and corresponding controls, hairpin miRNAs quantification kits, and U6 calibration real-time fluorescence quantitative PCR (RT-qPCR) kits were purchased from Genepharma Co. (Shanghai, China).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Negative Control, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Viruses

Article Title: High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine

doi: 10.3390/v16081346

Figure Lengend Snippet: Effect of skimmianine on HBV replication in HepG2.2.15 cells. HepG2.2.15 cells were seeded in 96-well plates, and 24 h later, skimmianine was added at 10 nM, 100 nM, 1 μM and 10 μM; entecavir at 200 nM; and DMSO at 2%. The medium was then replaced with fresh medium containing fresh compound and collected for the quantitation of the HBV-DNA level. On days 7, 10 and 13, the extracellular HBV-DNA level was quantitated by quantitative PCR and cell viability was analyzed on day 13 by a WST-8 assay. Each condition was tested using three wells of a 96-well plate. ( a ) HBV-DNA level in each condition. ( b ) HBV-DNA levels on day 10. Error bars show the standard deviation from the three wells. Each symbol shows the individual measurements. The differences in the means between 2% DMSO and each condition were analyzed with one-way analysis of variance with Bonferroni’s multiple comparisons test. **** p < 0.0001. ( c ) Cell viability relative to 2% DMSO. Each symbol shows the measurements. The differences in the means between 2% DMSO and each condition were analyzed with one-way analysis of variance with Bonferroni’s multiple comparisons test. ETV, entecavir; Skim, skimmianine; ns, not significant.

Article Snippet: Extracellular HBV-DNA levels were determined using an HBV Quantitative PCR (qPCR) Kit (Kubix, Hakusan, Japan) according to the manufacturer’s instructions.

Techniques: Quantitation Assay, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Viruses

Article Title: High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine

doi: 10.3390/v16081346

Figure Lengend Snippet: Inhibitory effect of skimmianine on viral entry for non-recombinant HBV. Non-recombinant HBV from genotype C, designated WT-HBV, to which the HiBiT coding sequence was not inserted, was used to infect PXB cells. Then, skimmianine at concentrations ranging from 10 pM to 100 nM was added in both the entry and replication protocols, as shown in a. The medium was replaced with fresh medium containing fresh skimmianine every 3 days, and then the amounts of HBsAg ( a ) and HBV-DNA ( b ) in the medium were quantitated by ELISA and quantitative PCR, respectively, on day 21 after infection. Each symbol shows the individual measurements. Differences in the means between DMSO treatment and each treatment were analyzed via a one-way analysis of variance test. * p < 0.05, ** p < 0.01, and **** p < 0.0001. ns, not significant.

Article Snippet: Extracellular HBV-DNA levels were determined using an HBV Quantitative PCR (qPCR) Kit (Kubix, Hakusan, Japan) according to the manufacturer’s instructions.

Techniques: Recombinant, Sequencing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Infection

Journal: Viruses

Article Title: High-Throughput Screening of Antiviral Compounds Using a Recombinant Hepatitis B Virus and Identification of a Possible Infection Inhibitor, Skimmianine

doi: 10.3390/v16081346

Figure Lengend Snippet: Effect of skimmianine on attachment and internalization. HepG2-NTCP-YFP cells or PXB cells were pre-chilled on ice for 15 min and then incubated with 20,000 gEq/cell HBV derived from HepAD38 cells at 4 °C for 1.5 h either with indicated concentrations of skimmianine or without skimmianine (DMSO 2%). Heparin was used as a negative control at a final concentration of 100 IU/mL. After incubation, for attachment assay, cells were vigorously washed 3 times to remove free HBV. For internalization assay, cells were cultured at 37 °C for 6 h, and then they were trypsinized and washed 3 times to remove cell-attached HBV. Each DNA sample was extracted and quantified by quantitative PCR and normalized to 100 ng total DNA. ( a ) The results from HepG2-NTCP-YFP. ( b ) Those from PXB cells. Each symbol shows the individual measurements. Differences in the means between DMSO treatment and each treatment were analyzed via a one-way analysis of variance test. **** p < 0.0001. ns, not significant.

Article Snippet: Extracellular HBV-DNA levels were determined using an HBV Quantitative PCR (qPCR) Kit (Kubix, Hakusan, Japan) according to the manufacturer’s instructions.

Techniques: Incubation, Derivative Assay, Negative Control, Concentration Assay, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Viruses

Article Title: Transcriptome Analysis in Air–Liquid Interface Porcine Respiratory Epithelial Cell Cultures Reveals That the Betacoronavirus Porcine Encephalomyelitis Hemagglutinating Virus Induces a Robust Interferon Response to Infection

doi: 10.3390/v16060939

Figure Lengend Snippet: Air–liquid interface porcine respiratory epithelial cells (ALI-PREC) susceptibility toward porcine hemagglutinating encephalomyelitis virus (PHEV) infection. ( A – D ) Completely differentiated ALI-PRECs (day 30) treated with infection medium only, i.e., without virus (mock-inoculated) ( A ), ( B ) with HA (titer of 128) of PHEV 67 N for 24 h post-inoculation (hpi), ( C ) 36 hpi, and ( D ) 48 hpi. Bar, 100 μm. Representative images from two biological and three technical replicates. ( E ) Detection of PHEV nucleocapsid gene using reverse transcription-qPCR. RNA from the subnatants collected from ALI-PRECs treated with PHEV was analyzed using RT-qPCR developed by ISU and Tetracore. Collection time is shown in hours. A sample volume of 5 μL of extracted sample RNA along with internal control was added to the qPCR master mix. All qPCRs were performed with a negative extraction control (NEC), a positive extraction control (PEC), and a no-template control (NTC) included in each run. Samples from two biological replicates and three technical replicates. Statistical analysis was performed using Fisher’s LSD multiple-comparison test (GraphPad Prism 9.0.1). **, p value < 0.01, and ****, p value < 0.0001.

Article Snippet: A quantitative PHEV N gene-based RT-PCR (RT-qPCR) developed by Tetracore (Tetracore, Inc., Rockville, MD, USA) and Iowa State University [ ] was used to confirm and quantify PHEV infection in vivo (CDCD pigs) and ex vivo (ALI-PRECs cultures).

Techniques: Virus, Infection, Reverse Transcription, Quantitative RT-PCR, Control, Extraction, Comparison

Journal: Viruses

Article Title: Transcriptome Analysis in Air–Liquid Interface Porcine Respiratory Epithelial Cell Cultures Reveals That the Betacoronavirus Porcine Encephalomyelitis Hemagglutinating Virus Induces a Robust Interferon Response to Infection

doi: 10.3390/v16060939

Figure Lengend Snippet: Quantitative PCR analysis of ALI-PRECs inoculated with PHEV or mock inoculated with infection medium. Bar graph showing relative quantification (RQ) levels of MDA5 ( A ), STAT1 ( B ), Mx1 ( C ), CXCL10 ( D ), and CCL5 ( E ) measured in ALI-PRECs treated with HA (titer of 128) of PHEV and mock inoculum at respective h post-infection (x axes). RQ values were calculated using the 2−ΔΔCT method. The data are normalized against the geometric mean for three endogenous control genes ( EIF3K , PPIA , and RPL10 ). This graph is generated from three technical replicates and two biological replicates (2 pigs). Statistical analysis was performed using Fisher’s LSD multiple-comparison test (GraphPad Prism 9.0.1). *, p value < 0.05; **, p value < 0.001; ***, p value < 0.005; ****, p value < 0.0001.

Article Snippet: A quantitative PHEV N gene-based RT-PCR (RT-qPCR) developed by Tetracore (Tetracore, Inc., Rockville, MD, USA) and Iowa State University [ ] was used to confirm and quantify PHEV infection in vivo (CDCD pigs) and ex vivo (ALI-PRECs cultures).

Techniques: Real-time Polymerase Chain Reaction, Infection, Control, Generated, Comparison

Journal: Oncology Letters

Article Title: DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

doi: 10.3892/ol.2021.13129

Figure Lengend Snippet: DNMT3B binds to and methylates the miR-152-3p core region. (A) Bisulfite treated DNA samples from A549, A549/DDP and A549/ADM cells were amplified with methylated and non-methylated primers at the same time, and the amplification level was detected by PCR. (B) Bisulfite sequencing was used to detect the methylation level of miR-152-3p in A549, A549/DDP and A549/ADM cells. Five respective clones from each group were sequenced. (C) ChIP assay detected the binding of DNMT3B, DNMT3A and DNMT1 proteins to the core region of miR-152-3p. (D) Bisulfite sequencing results of the methylation levels in the core region of miR-152-3p in A549 cells were detected after treatment with DNMT3B/DNMT1 methylase inhibitors in three independent experiments. (E) Relative mRNA expression levels of miR-152-3p and NCAM1 in A549 cells treated with DNMT3B methylase inhibitor. Two-tailed Student's t-test was used for statistical analysis. *P<0.05, ***P<0.001. DNMT, DNA methyltransferase; miR, microRNA; IP, immunoprecipitation, ChIP, chromatin IP; NCAM1, neural cell adhesion molecule 1; U, unmethylated; M, methylated.

Article Snippet: Target gene mRNA levels were examined using a fluorescent quantitative PCR kit (PerfectStart Green qPCR SuperMix; Beijing Transgen Biotech Co., Ltd.).

Techniques: Amplification, Methylation, Methylation Sequencing, Clone Assay, Binding Assay, Expressing, Two Tailed Test, Immunoprecipitation, Chromatin Immunoprecipitation

Journal: Oncology Letters

Article Title: DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

doi: 10.3892/ol.2021.13129

Figure Lengend Snippet: miR-152-3p targets and inhibits NCAM1. (A) Reverse transcription-quantitative PCR demonstrated the effects of miR-152-3p mimics and inhibitor on NCAM1 transcription level in A549 cells. (B) Western blotting revealed the effects of miR-152-3p mimics and inhibitor on NCAM1 protein levels in A549 cells. One-way ANOVA was used for statistical analysis. (C) Relative luciferase activity in the indicated groups from three independent tests. Two-tailed Student's t-test was used for statistical analysis. (D) Relative mRNA expression of miR-152-3p and NCAM1 in A549 and A549/DDP cells. (E) Relative expression of miR-152-3p in lung squamous cell carcinoma samples. Group A referred to the expression of miR-152-3p in 30 patients with LUSC and group B to that of 44 adjacent normal tissues. (F) Relative mRNA expression of NCAM1 in LUAD/LUSC and normal tissues from The Cancer Genome Atlas dataset. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 as indicated or vs. A549. miR, microRNA; NC, negative control; NCAM1, neural cell adhesion molecule 1; WT, wild type; MUT, mutant; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; T, tumor; N, normal.

Article Snippet: Target gene mRNA levels were examined using a fluorescent quantitative PCR kit (PerfectStart Green qPCR SuperMix; Beijing Transgen Biotech Co., Ltd.).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Luciferase, Activity Assay, Two Tailed Test, Expressing, Negative Control, Mutagenesis

Journal: Oncology Letters

Article Title: DNMT3B regulates proliferation of A549 cells through the microRNA-152-3p/NCAM1 pathway

doi: 10.3892/ol.2021.13129

Figure Lengend Snippet: Knockdown of DNMT3B increases miR-152-3p expression and delays cell proliferation. (A) Cell survival rate of A549 cell lines transfected with sh-DNMT3B and or miR-152-3p inhibitor, as indicated by the MTT assay. (B) Colony formation assay was used to detect the proliferative ability of the indicated groups. (C) Reverse transcription-quantitative PCR demonstrated the different expression levels of miR-152-3p and NCAM1 in the indicated groups. (D) Expression levels of NCAM1, cleaved-caspase-3 and cleaved-PARP proteins in A549 cells detected via western blotting. One-way ANOVA was used for statistical analysis. *P<0.05, **P<0.01, ***P<0.001. DNMT, DNA methyltransferase; miR, microRNA; NC, negative control; NCAM1, neural cell adhesion molecule 1; sh, short hairpin.

Article Snippet: Target gene mRNA levels were examined using a fluorescent quantitative PCR kit (PerfectStart Green qPCR SuperMix; Beijing Transgen Biotech Co., Ltd.).

Techniques: Knockdown, Expressing, Transfection, MTT Assay, Colony Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Negative Control