Journal: International journal of oncology

Article Title: Extracellular domain of semaphorin 5A serves a tumor‑suppressing role by activating interferon signaling pathways in lung adenocarcinoma cells.

doi: 10.3892/ijo.2022.5311

Figure Lengend Snippet: Figure 4. Expression levels of tumor suppressor genes in the interferon signaling pathway are upregulated by different SEMA5A domains. (A) Schematic depiction of the canonical pathway of interferon signaling, colored according to gene expression in the presence of 5A‑Full. The darkness of red color indicates the fold change of each gene. (B) Schematic depiction of the canonical pathway of interferon signaling, colored according to gene expression in the presence of 5A‑ECD. (C) Schematic depiction of the canonical pathway of interferon signaling, colored according to gene expression in the presence of 5A‑ICD. (D) Relative expression levels of JAK1, JAK2 and TYK2 examined by RT‑qPCR in A549 cells overexpressing different SEMA5A domains. (E) Relative expression levels of JAK1, JAK2 and TYK2 examined by RT‑qPCR in H1299 cells overexpressing different SEMA5A domains. (F) Relative expression levels of STAT1, STAT2 and IRF9 in A549 cells overexpressing different SEMA5A domains. (G) Relative expression levels of STAT1, STAT2 and IRF9 in H1299 cells overexpressing different SEMA5A domains. (H) Relative expression levels of IFIT1, G1P2, G1P3, IFITM1, IFITM2 and IFITM3 in A549 cells overexpressing different SEMA5A domains. (I) Relative expression levels of IFIT1, G1P2, G1P3, IFITM1, IFITM2 and IFITM3 in H1299 cells overexpressing different SEMA5A domains. GAPDH: internal control. Experiments were repeated in triplicate, and the results are presented as the mean ± SD. One‑way ANOVA and Bonferroni post hoc test were performed. *P<0.05 vs. empty control.

Article Snippet: The membranes were blocked with the undiluted Lightning Blocking Buffer (Enginelife Science Co., Ltd.) for 10 min at room temperature and incubated with the following primary antibodies overnight at 4 ̊C: SEMA5A (dilution, 1:1,000; cat. no. PAL924Hu01; Cloud‐Clone Corp.), p‐STAT1 (dilution, 1:1,000; cat. no. AP0109; ABclonal Biotech Co., Ltd.), STAT1 (dilution, 1:1,000; cat. no. 10144‐2‐AP; ProteinTech Group, Inc.), p‐STAT2 (dilution, 1:1,000; cat. no. AP0284; ABclonal Biotech Co., Ltd.), STAT2 (dilution, 1:1,000; cat. no. 72604; Cell Signaling Technology, Inc.), p‐JAK1 (dilution, 1:1,000; cat. no. AP0530; ABclonal Biotech Co., Ltd.), JAK1 (dilution, 1:1,000; cat. no. 3332; Cell Signaling Technology, Inc.), p‐JAK2 (dilution, 1:1,000; cat. no. AP0531; ABclonal Biotech Co., Ltd.), JAK2 (dilution, 1:1,000; cat. no. 3230; Cell Signaling Technology, Inc.), IFIT1 (dilution, 1:1,000; cat. no. 14769; Cell Signaling Technology, Inc.), G1P2 (ISG15) (dilution, 1:1,000; cat. no. 2758; Cell Signaling Technology, Inc.), ACTB (dilution, 1:5,000; cat. no. 3700; Cell Signaling Technology, Inc.), caspase‐3 (CASP3) (dilution, 1:1,000; cat. no. 9662; Cell Signaling Technology, Inc.), cleaved caspase‐3 (dilution, 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.) and GAPDH (dilution, 1:5,000; cat. no. 10494‐1‐AP; ProteinTech Group, Inc.).

Techniques: Expressing, Gene Expression, Control

Journal: International journal of oncology

Article Title: Extracellular domain of semaphorin 5A serves a tumor‑suppressing role by activating interferon signaling pathways in lung adenocarcinoma cells.

doi: 10.3892/ijo.2022.5311

Figure Lengend Snippet: Figure 5. Overexpression of 5A‑Full and 5A‑ECD increases the amount of phosphorylated STAT1 in A549 cells. (A) Western blotting of total lysates from A549 cells overexpressing 5A‑Full. (B) Western blotting of total lysates from H1299 cells overexpressing 5A‑Full or 5A‑ECD. (C) Semi‑quantification of SEMA5A from the conditions of (A). (D) Semi‑quantification of SEMA5A from the conditions of (B). (E) Relative protein expression levels of differentially expressed genes examined by western blotting in A549 cells. GAPDH or ACTB was used as the loading control. (F) Semi‑quantification of p‑STAT1, p‑STAT2, p‑JAK1, p‑JAK2, IFIT1 and G1P2 in A549 cells. Each phosphorylated protein was normalized to its respective total protein, i.e., p‑STAT1 was normalized to t‑STAT1, p‑STAT2 to t‑STAT2, p‑JAK1 to t‑JAK1 and p‑JAK2 to t‑JAK2. IFIT1 and G1P2 were normalized to ACTB. (G) Relative protein expression levels of differ‑ entially expressed genes examined by western blotting in H1299 cells. ACTB was used as the loading control. (H) Semi‑quantification of p‑STAT1, IFIT1 and G1P2 in H1299 cells. Each phosphorylated protein was normalized to its respective total protein, i.e., p‑STAT1 was normalized to t‑STAT1, p‑STAT2 to t‑STAT2, p‑JAK1 to t‑JAK1 and p‑JAK2 to t‑JAK2. IFIT1 and G1P2 were normalized to ACTB. Experiments were repeated in triplicate, and the results are presented as the mean ± SD. One‑way ANOVA and Bonferroni post hoc test were performed. *P<0.05 vs. Empty. 5A‑ECD, SEMA5A extracellular domain; 5A‑Full, SEMA5A full length; ACTB, actin β; G1P2, glycogenin 1 pseudogene 2; IFIT1, interferon induced protein with tetratricopeptide repeats 1; JAK, Janus kinase; p, phosphorylated; SEMA5A, semaphorin 5A; t, total.

Article Snippet: The membranes were blocked with the undiluted Lightning Blocking Buffer (Enginelife Science Co., Ltd.) for 10 min at room temperature and incubated with the following primary antibodies overnight at 4 ̊C: SEMA5A (dilution, 1:1,000; cat. no. PAL924Hu01; Cloud‐Clone Corp.), p‐STAT1 (dilution, 1:1,000; cat. no. AP0109; ABclonal Biotech Co., Ltd.), STAT1 (dilution, 1:1,000; cat. no. 10144‐2‐AP; ProteinTech Group, Inc.), p‐STAT2 (dilution, 1:1,000; cat. no. AP0284; ABclonal Biotech Co., Ltd.), STAT2 (dilution, 1:1,000; cat. no. 72604; Cell Signaling Technology, Inc.), p‐JAK1 (dilution, 1:1,000; cat. no. AP0530; ABclonal Biotech Co., Ltd.), JAK1 (dilution, 1:1,000; cat. no. 3332; Cell Signaling Technology, Inc.), p‐JAK2 (dilution, 1:1,000; cat. no. AP0531; ABclonal Biotech Co., Ltd.), JAK2 (dilution, 1:1,000; cat. no. 3230; Cell Signaling Technology, Inc.), IFIT1 (dilution, 1:1,000; cat. no. 14769; Cell Signaling Technology, Inc.), G1P2 (ISG15) (dilution, 1:1,000; cat. no. 2758; Cell Signaling Technology, Inc.), ACTB (dilution, 1:5,000; cat. no. 3700; Cell Signaling Technology, Inc.), caspase‐3 (CASP3) (dilution, 1:1,000; cat. no. 9662; Cell Signaling Technology, Inc.), cleaved caspase‐3 (dilution, 1:1,000; cat. no. 9661; Cell Signaling Technology, Inc.) and GAPDH (dilution, 1:5,000; cat. no. 10494‐1‐AP; ProteinTech Group, Inc.).

Techniques: Over Expression, Western Blot, Expressing, Control

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: (A) Dapivirine; (B) Etavirine; (C) Rilpivirine;( D) EC50 of the three NNTIs against A/California/07/2009 and B/Brisbane/60/2008 and CC50 of the three NNTIs with a 48 h incubation time with MDCK cell line. The EC50 and CC50 values are the mean of two independent experiments ± standard deviation.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: Incubation, Standard Deviation

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: MDCK cells were infected with the A/WSN/33 (H1N1) virus at −2 h time point; viruses were first incubated at 4 °C for 1 h for attachment followed by 37 °C for 1 h for viral entry. At time point 0 h, cells were washed with PBS buffer and viruses were harvested at 12 h p.i. The titer of harvested virus was determined by plaque assay. Arrows indicate the period in which (A) 1 μM oseltamivir carboxylate or (B) 3 μM dapivirine was present. Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of viral titer is the mean of two independent experiments ± standard deviation.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: Infection, Virus, Incubation, Plaque Assay, Comparison, Control, Standard Deviation

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: EC 50 of dapivirine against a panel of influenza A and B viruses and selection index of dapivirine with MDCK and A549 cell lines.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: Selection

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: (A) CC50 of dapivirine against MDCK and A549 cell lines with a 48 h incubation time; The CC50 values are the mean of two independent experiments ± standard deviation. (B) CC50 of dapivirine against MDCK, HEK 293 or A549 cell lines with a 24 h incubation time. The concentration at 3 μM (Figure A) and 10 μM (Figure B) were indicated with arrows. The CC50 values are the mean of two independent experiments ± standard deviation. (C) HEK293 or MDCK cells were infected with A/California/7/2009 (H1N1) at MOI of 0.01; 3 μM dapivirine or 1 μM oseltamivir carboxylate was added after viral infection. Viruses from supernatant were harvested 24 h p.i. and titers were determined by plaque assay. *** P < 0.001. The value of viral titer is the mean of two independent experiments ± standard deviation. (D) A549 cells were infected with A/WSN/1933 (H1N1) at MOI of 0.01; 3 μM dapivirine or 1 μM oseltamivir carboxylate was added after viral infection. Viruses from supernatant were harvested 24 h p.i. and the titers were determined by plaque assay. Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of viral titer is the mean of two independent experiments ± standard deviation. (E) A549 cells were infected with A/WSN/1933 (H1N1) at MOI of 0.01; DMSO (control) or 3 μM dapivirine was added after infection. Viruses from supernatant were harvested 2, 4, 6, 8, 12, 24, 48, 72, or 96 h post infection. Virus titers were determined by plaque assay.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: Incubation, Standard Deviation, Concentration Assay, Infection, Plaque Assay, Comparison, Control, Virus

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: Serial viral passage experiments with dapivirine drug selection pressure.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: Selection

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: (A) Retention of influenza vRNPs in cytoplasm by dapivirine. A549 cells infected with the A/WSN/33 (H1N1) virus (MOI = 30) were treated with DMSO or 30 μM of dapivirine. After fixation at indicated times (2, 4, and 6 h p.i.), cells were stained with mouse anti-influenza A NP antibody, rabbit anti-M1, and DAPI to determine the viral NP, M1 protein, and nucleus, respectively. (B) CC50 of dapivirine against A549 cell line with a 6 h incubation time. The concentration at 30 μM was indicated with an arrow. The CC50 value is the mean of two independent experiments ± standard deviation. (C–F) Dapivirine reduced the levels of viral RNAs and proteins. A549 cells infected with the A/WSN/33 (H1N1) virus (MOI = 1) were treated with DMSO and 10 or 30 μM of dapivirine. At 6 h p.i., cells were harvested and total viral RNA was extracted and quantified by RT-qPCR (C and D). Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of RNA level is the mean of two independent experiments ± standard deviation. Viral protein levels were quantified by western blot (E) and immunofluorescence (F). (G–I) Mini-genome assay. A549 cells were transfected with a combination of plasmids for minigenome assay (see in Materials and methods). Dapivirine was added to the culture medium at 2 h post transfection. After 22 h incubation, cells were harvest and the activities of Firefly (G) and Renilla (H) luciferases were measured using the dual luciferease kit from Promega. Renilla was served as an internal control to normalize the transfection efficiency (I). Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of luciferase signal is the mean of two independent experiments ± standard deviation.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: Infection, Virus, Staining, Incubation, Concentration Assay, Standard Deviation, Quantitative RT-PCR, Comparison, Control, Western Blot, Immunofluorescence, Transfection, Luciferase

Journal: Antiviral research

Article Title: Discovery of dapivirine, a Nonnucleoside HIV-1 Reverse Transcriptase Inhibitor, as a Broad-Spectrum Antiviral against Both Influenza A and B Viruses

doi: 10.1016/j.antiviral.2017.07.016

Figure Lengend Snippet: Antiviral results of combinational treatments.

Article Snippet: Compounds, cell lines, viruses, and viral infection Dapivirine (Cat # S2914), etravirine (Cat # S3080), and rilpivirine (Cat # S7303) were ordered from Selleckchem.

Techniques: