Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: Expression of circFNDC3B in gastric cancer cells. (a) The level of circFNDC3B was analyzed by qRT‐PCR in control cells from the gastric epithelium cell line GES‐1, as well as in the AGS, SGC‐7901, BCG‐823 and MGC‐803 gastric cancer cell lines. (b) The expression level of FNDC3B mRNA was analyzed by qRT‐PCR in control cells from the gastric epithelium cell line GES‐1, as well as in the AGS, SGC‐7901, BCG‐823, and MGC‐803 gastric cancer cell lines. Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.0001. FNDC3B: fibronectin type III domain‐containing protein 3B; qRT‐PCR: quantitative reverse transcription‐PCR; SEM : standard error of the mean

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Reverse Transcription

Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: CircFNDC3B did not influence FNDC3B expression. (a) The pcDNA3.1‐circFNDC3B‐mini vector was constructed without a restriction enzyme site by Gibson Assembly DNA clone technology. The light green arrows show the inverted repeat supporting circFNDC3B circularization. Pink triangle primers or blue triangle primers were designed to amplify vector or circFNDC3B exons 5 and 6. (b) The circFNDC3B expression vector successfully induced an increase in circFNDC3B expression in MGC‐803 cells (EV: empty vector; OVER: overexpressing circFNDC3B; WT: wild‐type). (c) The three siRNAs targeting the circFNDC3B junction sequence were designed and only exhibited the sense strand. (d) BGC‐823 cells were transfected with three siRNAs specifically targeting circFNDC3B (S1, S2, S3) or a negative control siRNA for 48 hr, and the level of silencing circFNDC3B was detected by qRT‐PCR (WT: wild‐type; NC: negative control; siRNA: S1, S2, S3). (e,g) MGC‐803 cells were transfected with the circFNDC3B vector or an empty vector. Cells were cultured for 48 hr, and then we examined FNDC3B mRNA and protein expression by qRT‐PCR and western blot. The results showed that overexpression of circFNDC3B did not significantly increase the mRNA or protein level of FNDC3B (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B). (f,h) BGC‐823 cells were transfected with siRNA (S1, S2, S3) or a negative control siRNA. Cells were cultured for 48 hr, and then we examined circFNDC3B and FNDC3B mRNA and protein expression by qRT‐PCR and western blot, respectively. The silencing of circFNDC3B with S1 and S2 did not statistically affect FNDC3B mRNA or protein levels (WT: wild‐type; NC: negative control; siRNA: S1, S2, S3). (i,j) MGC‐803 cells were transfected with the pEGFP‐FNDC3B vector or an empty vector. After 48 hr, FNDC3B levels and circFNDC3B levels were detected by western blot and qRT‐PCR. The results suggest that FNDC3B mRNA increased circFNDC3B (WT: wild‐type; EV: empty vector against EGFP‐FNDC3B; FNDC3B mRNA: overexpressing FNDC3B mRNA). Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.0001. FNDC3B: fibronectin type III domain‐containing protein 3B; qRT‐PCR: quantitative reverse transcription‐PCR; SEM : standard error of the mean; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Plasmid Preparation, Construct, Sequencing, Transfection, Negative Control, Quantitative RT-PCR, Cell Culture, Western Blot, Over Expression, Reverse Transcription, Small Interfering RNA

Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: CircFNDC3B promoted migration and invasion. (a,b) A wound‐healing assay was performed to evaluate the migration ability of MGC‐803 and BGC‐823. MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. A line was drawn from one end of the well to the other. The wound‐healing process was recorded by microscopy at 100× magnification. The red line indicates the measured distance after cell migration (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (c,d) The migration ability was evaluated by the red line distance of the wound‐healing gap. Quantitative results showed that the wound gap was obviously shortened in MGC‐803 cells transfected with the circFNDC3B vector and that silencing circFNDC3B suppressed cell migration in BGC‐823 cells (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (e,f) Transwell assays were performed to evaluate the invasion ability of MGC‐803 and BGC‐823 cells after overexpression and silencing of circFNDC3B. MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. The cells were trypsinized, resuspended, and seeded on the upper chamber of well‐coated ECM gel. After 24 hr, the invaded cells on the bottom of the well were fixed and stained. The invaded cells stained by crystal violet were calculated as the degree of invasion. Three different areas of each well were randomly selected and observed under a microscope at 200× magnification. The results showed that MGC‐803 cells transfected with the circFNDC3B vector invaded more than the empty vector cells, whereas silencing circFNDC3B in BGC‐823 cells caused them to invade less than BGC‐823 cells with negative control (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (g,h) Quantitative results showed that overexpressing circFNDC3B promoted cell invasion in MGC‐803 cells and that silencing circFNDC3B suppressed cell invasion in BGC‐823 cells (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (i,j) BGC‐823 cells were transfected with the circFNDC3B vector, and results showed that circFNDC3B indeed promoted cell migration and invasion (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B). Finally, we validated that circFNDC3B could promote cell migration and invasion. Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.0001. ECM: extracellular matrix; FNDC3B: fibronectin type III domain‐containing protein 3B; SEM : standard error of the mean; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Migration, Wound Healing Assay, Transfection, Plasmid Preparation, Negative Control, Microscopy, Over Expression, Staining, Small Interfering RNA

Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: CircFNEC3B reduced the expression of E‐cadherin. (a) MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. Overexpression of circFNDC3B only inhibited the expression of E‐cadherin protein. (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B). (b) BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. Silencing of circFNDC3B upregulated the expression of E‐cadherin and inhibited the expression of N‐cadherin, Vimentin, and SNAI1 in protein. (WT: wild‐type; NC: negative control; siRNA: S1, S2) (c,d) MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. qRT‐PCR verified that circFNDC3B did not affect the expression of ECAD (CDH1) mRNA, SNAI1 mRNA, VIM mRNA, and NCAD (CDH2) mRNA. (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.0001. FNDC3B: fibronectin type III domain‐containing protein 3B; qRT‐PCR: quantitative reverse transcription‐PCR; SEM : standard error of the mean; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Negative Control, Quantitative RT-PCR, Reverse Transcription, Small Interfering RNA

Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: CircFNDC3B interacted with IGF2BP3 and CD44 mRNA. (a) The expression level of IGF2BP3 was evaluated in MGC‐803 and BGC‐823 cells by western blot. MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. The results suggested that circFNDC3B did not significantly influence IGF2BP3 (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (b) Ectopic expression or silencing of circFNDC3B did not statistically influence IGF2BP3 mRNA expression by qRT‐PCR (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (c) RIP assay using an antibody against IGF2BP3 was performed to evaluate the interaction between IGF2BP3, circFNDC3B, and CD44 mRNA in MGC‐803 and BGC‐823 cell lines. CircFNDC3B or CD44 mRNA was amplified by cirdFNDC3B primers or CD44 primers. The results showed that IGF2BP3 could interact with circFNDC3B and CD44 mRNA by RT‐PCR (INPUT: total RNA was reverse‐transcribed with circFNDC3B primers or CD44 mRNA primers. IgG and IGF2BP3: after incubation with IgG or IGF3BP3 antibody). (d) RNA pull‐down was performed to assess the role of circFNDC3B between IGF2BP3 and CD44 mRNA by the circFNDC3B biotinylated probe. Western blot detected the interaction between circFNDC3B and IGF2BP3. RT‐PCR assessed the interaction between circFNDC3B and CD44 mRNA (INPUT: total protein or total RNA were incubated with IGF2BP3 antibody or reverse‐transcribed and amplified CD44 primers, scrambled and circFNDC3B: after incubation with biotin‐labeled scrambled oligonucleotide or circFNDC3B oligonucleotide). These results suggested that circFNDC3B could interact with IGF2BP3 and CD44 mRNA. Finally, RIP and RNA pull‐down assays validated the ternary complex of circFNDC3B‐IGF2BP3‐ CD44 mRNA. (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). FNDC3B: fibronectin type III domain‐containing protein 3B; RIP: RNA immunoprecipitation; RT‐PCR: reverse transcription‐PCR; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, Negative Control, Quantitative RT-PCR, Amplification, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Incubation, Labeling, RNA Immunoprecipitation, Small Interfering RNA

Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: CircFNDC3B increased CD44 levels by binding to IGF2BP3. (a) The level of CD44 mRNA in MGC‐803 and BGC‐823 cells was evaluated by qRT‐PCR. MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) for 48 hr. BGC‐823 cells were transfected with siRNA (S1, S2) or negative control siRNA (NC) for 48 hr. The results suggested that ectopic expression of circFNDC3B increased CD44 mRNA, whereas silencing of circFNDC3B reduced CD44 mRNA (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B; NC: negative control; siRNA: S1, S2). (b,c) Western blot was performed to evaluate CD44 protein in MGC‐803 and BGC‐823 cells. The gray scale quantitative results suggested ectopic expression of circFNDC3B remarkably upregulated CD44 levels, and inversely, silencing of circFNDC3B downregulated CD44 levels. (d) We also overexpressed circFNDC3B on BGC‐823 cells, and the results showed that overexpression of circFNDC3B increased CD44 expression (WT: wild‐type; EV: empty vector; OVER: overexpressing circFNDC3B). (e) We detected the role of IGF2BP3 between circFNDC3B and CD44 mRNA. MGC‐803 cells were transfected with siRNA against IGF2BP3 (S1, S2, S3) to detect the level of silencing IGF2BP3 and CD44 expression by Western blot. The results showed that silencing IGF2BP3 obviously reduced CD44 expression (WT: wild‐type; NC‐IGF2BP3: negative control; S1‐IGF2BP3, S2‐IGF2BP3, S3‐IGF2BP3: siRNA targeting IGF2BP3). (f) Then, MGC‐803 cells were transfected with siRNA against IGF2BP3 and transfected with the circFNDC3B vector again after 48 hr. Western blotting was performed to detect CD44 levels. The gray scale quantitative results suggested that when IGF2BP3 was silenced in MGC‐803 cells, overexpression of circFNDC3B did not truly increase the expression of CD44 (WT: wild‐type; NC+EV: negative control siRNA and empty vector; S1+OVER, S2+OVER, S3+OVER: siRNA against IGF2BP3 and circFNDC3B vector). (g) qRT‐PCR results showed that IGF2BP3 mRNA and CD44 mRNA and circFNDC3B levels were regulated (WT: wild‐type; NC+EV: negative control siRNA and empty vector; S1+OVER, S2+OVER, S3+OVER: siRNA against IGF2BP3 and circFNDC3B vector). Data were expressed as the mean ± SEM and were analyzed by independent samples t test, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.0001. FNDC3B: fibronectin type III domain‐containing protein 3B; qRT‐PCR: quantitative reverse transcription‐PCR; SEM : standard error of the mean; siRNA: small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Binding Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Negative Control, Expressing, Western Blot, Over Expression, Reverse Transcription, Small Interfering RNA

Journal: Journal of Cellular Physiology

Article Title: FNDC3B circular RNA promotes the migration and invasion of gastric cancer cells via the regulation of E‐cadherin and CD44 expression

doi: 10.1002/jcp.28588

Figure Lengend Snippet: CircFNDC3B has translational protein activity. (a) The figure shows start codon and stop codon positions, 2r+8 rolling circle translation format, and protein sequence information (blue ring: 5 exon; orange ring: 6 exon; black square: junction; blue square: start codon; orange square: stop codon). (b) The circFNDC3B translational protein was detected by inserting a 3× flag–tag at both ends of the initiation codon and the stop codon of the circFNDC3B cyclization vector pcDNA3.1 circFNDC3B mini vector. MGC‐803 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) or vector with flag–tag for 48 hr. BGC‐823 cells were transfected with circFNDC3B vector (OVER) or empty vector (EV) or vector with flag–tag for 48 hr. Western blot results showed that circFNDC3B could translate approximately 25 kD peptide against the FNDC3B‐specific antibody and could also detect the 150 and 70 kD FNDC3B variants (purple square: 3× flag–tag; blue ring: 5 exon; orange ring: 6 exon; black square: junction; blue square: start codon; orange square: stop codon). (c) A linear expression vector was synthesized and constructed according to the circFNDC3B rolling circle translation sequence. MGC‐803 cells were transfected with p3× flag‐CMV‐10‐circFNDC3B or p3× flag‐CMV‐10 empty vector (EV) for 48 hr. Western blot results showed that circFNDC3B has translational protein activity against flag–tag antibodies (purple square: 3× flag; blue ring: 5 exon; orange ring: 6 exon; black square: junction; blue square: start codon; orange square: stop codon). FNDC3B: fibronectin type III domain‐containing protein 3B [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China).

Techniques: Activity Assay, Sequencing, FLAG-tag, Plasmid Preparation, Transfection, Western Blot, Expressing, Synthesized, Construct

Journal: Experimental and Therapeutic Medicine

Article Title: NICE-3-knockdown induces cell cycle arrest and autophagy in lung adenocarcinoma cells via the AKT/mTORC1 signaling pathway

doi: 10.3892/etm.2021.10057

Figure Lengend Snippet: NICE-3-knockdown inhibits cellular proliferation, colony formation and the cell cycle. (A) A549 cells were transfected with si-NICE-3 for 48 h. NICE-3 protein expression was then assessed by western blotting, indicating that si-NICE-3-#3 exerted the greatest inhibitory effect. (B) A549 and H1993 cells were transfected with si-NICE-3 and proliferation was assessed by cell counting. NICE-3-knockdown suppressed the proliferation of A549 and H1993 cells. (C) Colony formation assay results of A549 and H1993 cells transfected with si-NICE-3 or si-control. (D) Cell cycle analysis results of A549 cells transfected with si-NICE-3 or si-control. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001. si, small interfering RNA.

Article Snippet: For autophagy assay, A549 and H1993 cells were transfected with si-NICE-3 or si-control for 48 h before being treated with the autophagy inhibitor bafilomycin A1 (100 nM; Targetmol) at room temperature for 1 h, followed by western blotting assay.

Techniques: Knockdown, Transfection, Expressing, Western Blot, Cell Counting, Colony Assay, Control, Cell Cycle Assay, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: NICE-3-knockdown induces cell cycle arrest and autophagy in lung adenocarcinoma cells via the AKT/mTORC1 signaling pathway

doi: 10.3892/etm.2021.10057

Figure Lengend Snippet: NICE-3-knockdown suppresses the migration and invasion of lung adenocarcinoma cells. Migration and invasion of A549 and H1993 cells transfected with si-NICE-3 or si-control were assessed using Transwell assays without or with Matrigel, respectively. Representative images are shown (magnification, x200). * P<005; ** P<0.01; *** P<0.001; **** P<0.0001. si, small interfering RNA.

Article Snippet: For autophagy assay, A549 and H1993 cells were transfected with si-NICE-3 or si-control for 48 h before being treated with the autophagy inhibitor bafilomycin A1 (100 nM; Targetmol) at room temperature for 1 h, followed by western blotting assay.

Techniques: Knockdown, Migration, Transfection, Control, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: NICE-3-knockdown induces cell cycle arrest and autophagy in lung adenocarcinoma cells via the AKT/mTORC1 signaling pathway

doi: 10.3892/etm.2021.10057

Figure Lengend Snippet: NICE-3-knockdown induces autophagy by inhibiting the AKT/mTORC1 signaling pathway. A549 and H1993 cells were transfected with si-NICE-3 or si-control for 48 h, followed by bafilomycin treatment for 1 h. LC3 and p62 proteins, as well as AKT and S6K phosphorylation, were evaluated by western blot analysis. (A) Conversion of LC3-I to LC3-II (LC3-II/LC3-I ratio as an indicator) was enhanced, and p62 protein levels were decreased by si-NICE-3, indicating increased autophagy. Representative western blots are shown. The bands were quantified using ImageJ and normalized to the loading control. (B) Phosphorylation of AKT and S6K were suppressed by NICE-3-knockdown, indicating decreased AKT and mTORC1 activity. Representative western blots are shown. The bands were quantified using ImageJ and normalized to the loading control. * P<005; ** P<0.01; *** P<0.001. si, small interfering RNA; mTORC1, mechanistic target of rapamycin complex 1; Baf, bafilomycin; p, phosphorylated.

Article Snippet: For autophagy assay, A549 and H1993 cells were transfected with si-NICE-3 or si-control for 48 h before being treated with the autophagy inhibitor bafilomycin A1 (100 nM; Targetmol) at room temperature for 1 h, followed by western blotting assay.

Techniques: Knockdown, Transfection, Control, Phospho-proteomics, Western Blot, Activity Assay, Small Interfering RNA

Journal: Hepatology (Baltimore, Md.)

Article Title: Hepatitis C virus JFH-1 strain infection in chimpanzees is associated with low pathogenicity and emergence of an adaptive mutation.

doi: 10.1002/hep.22422

Figure Lengend Snippet: Fig. 1. Infection profiles and T cell immune responses in patient serum–inoculated and JFH-1cc–inoculated chimpanzees. (A) Chimpanzee CH10273 was inoculated with patient serum containing 9.6 106 copies of HCV. (B) Chimpanzee CH10274 was inoculated with JFH-1cc containing 1.4 107 copies of HCV. White arrows indicate the time points at which HCV sequences were determined. T cell proliferation assay results against HCV core and NS5a are shown as stimulation index (middle panel). IFN- responses against HCV core and NS5a proteins or overlapping peptide pools of core and NS3 are shown as SFU per 2.5 105 cells (bottom panel).

Article Snippet: Standard T-cell proliferation assay was performed as described previously.14 Cells were stimulated with recombinant HCV genotype 2a core or NS5a protein (Fitzgerald Industries Interna- nloaded from https://aasldpubs.onlinelibrary.w iley.com /doi/10.1002/hep.22422 by U niversity O f T he Philippines, W iley O nline L ibrary on [22/01/2024].

Techniques: Infection, Proliferation Assay

Journal: Oncotarget

Article Title: Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling

doi: 10.18632/oncotarget.23562

Figure Lengend Snippet: Structures of recombinant JFH1 viruses carrying NS5A of a genotype 1b strain were indicated. We generated 3 JFH-1-based recombinant viruses: JFH1/5ACon1 (carrying NS5A of the genotype 1b Con1 strain), JFH1/5ACon1/i-wt (containing wild-type amino acids in the ISDR), and JFH1/5ACon1/i-7mut (containing 7 patient-derived amino acid substitutions in the ISDR). An alignment of amino acids in the ISDRs used in this study was also indicated. Con1 has 1 amino acid substitution and 7mut has 7 amino acid substitutions compared with the wild-type ISDR.

Article Snippet: To assess the susceptibility to the NS5A inhibitor, Daclatasvir was obtained from ChemScene (Monmouth Junction, NJ, USA).

Techniques: Recombinant, Generated, Derivative Assay

Journal: Oncotarget

Article Title: Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling

doi: 10.18632/oncotarget.23562

Figure Lengend Snippet: ( A ) Huh-7.5.1 cells were transfected with the full-length RNAs of JFH1/5ACon1, JFH1/5ACon1/i-wt, and JFH1/5ACon1/i-7mut. At 48 h post-transfection, the cell lysates were immunoprecipitated with an anti-NS5A antibody, followed by immunoblotting analysis with an anti-core antibody. ( B ) Huh-7.5.1 cells were transfected with the full-length RNAs of JFH1/5ACon1 (5ACon1), JFH1/5ACon1/i-wt (i-wt), and JFH1/5ACon1/i-7mut (i-7mut). At 48 h post-transfection, the cells were stained with anti-NS5A and anti-core antibodies and analyzed by fluorescence microscopy. Red: NS5A; Green: core; Blue: DAPI. ( C ) Huh-7.5.1 cells were transfected with the indicated full-length RNAs. At 48 h post-transfection, the cells were stained with an anti-core antibody and BODIPY and analyzed by fluorescence microscopy. Red: core; Green: Lipid droplets; Blue: DAPI. ( D ) Huh-7.5.1 cells were transfected with the indicated full-length RNAs. At 48 h post-transfection, the cells were stained with an anti-NS5A antibody and BODIPY and analyzed by fluorescence microscopy. Red: NS5A; Green: Lipid droplets; Blue: DAPI.

Article Snippet: To assess the susceptibility to the NS5A inhibitor, Daclatasvir was obtained from ChemScene (Monmouth Junction, NJ, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Fluorescence, Microscopy

Journal: Oncotarget

Article Title: Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling

doi: 10.18632/oncotarget.23562

Figure Lengend Snippet: ( A ) HepaRG cells were transfected with the full-length RNAs of JFH1/5ACon1 and JFH1/5ACon1/i-7mut. At 48 h post-transfection, the cells were treated with IFN-α or diluent. After 30 min, the cell lysates were subjected to immunoblotting analysis with the indicated antibody. ( B ) HepaRG cells were transfected with NS5A expression vectors (5ACon1 or 5ACon1/i-7mut) or an empty vector. At 48 h post-transfection, the cells were treated with IFN-α. After 30 min, the cell lysates were immunoprecipitated with anti-STAT1, anti-STAT2, or anti-NS5A antibodies, followed by immunoblotting analysis with the indicated antibodies. ( C ) HepaRG cells were transfected with NS5A expression vectors (5ACon1 or 5ACon1/i-7mut). At 48 h post-transfection, the cells were treated with IFN-α. After 30 min, the cells were stained with the indicated antibodies and analyzed by fluorescence microscopy. Red: NS5A; Green: STAT1; Blue: DAPI.

Article Snippet: To assess the susceptibility to the NS5A inhibitor, Daclatasvir was obtained from ChemScene (Monmouth Junction, NJ, USA).

Techniques: Transfection, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation, Staining, Fluorescence, Microscopy

Journal: Hepatology (Baltimore, Md.)

Article Title: Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

doi: 10.1002/hep.30647

Figure Lengend Snippet: Pre‐existing A156T facilitated 1a(TN) escape from combination treatment. 1a(TN)N3 harboring A156T and D168E as well as the original 1a(TN) were subjected to mono or combination treatment with grazoprevir and elbasvir (A) or glecaprevir and pibrentasvir (B) or mono or triple treatment with voxilaprevir, velpatasvir, and sofosbuvir (C) at the indicated fold‐EC50 until viral escape, control, or suppression occurred (see Materials and Methods section). Viral control occurred for 1a(TN) under double treatment and for 1a(TN)N3 under triple treatment. Viral suppression occurred for 1a(TN) under triple treatment. (D) Sanger sequencing of DAA targets of escape variants revealed acquisition of additional RASs in NS3P (brown) and NS5A domain I (purple). a The RAS L31V in NS5A was detected following viral spread to most of the culture cells on day 45 (data not shown), following treatment termination on day 31.

Article Snippet: Percentage of HCV infected cells was determined by immunostaining with anti‐NS5A‐9E10 and anti‐Core‐C7‐50 (Enzo Life Sciences and Abcam).

Techniques: Sequencing

Journal: Hepatology (Baltimore, Md.)

Article Title: Evolutionary Pathways to Persistence of Highly Fit and Resistant Hepatitis C Virus Protease Inhibitor Escape Variants

doi: 10.1002/hep.30647

Figure Lengend Snippet: Analysis of HCV sequences of genotype 1a–infected patients failing DAA treatment and harboring A156G or V. Patients A, B, C, D, and E were treated with glecaprevir + piprentasvir (A,D), grazoprevir + elbasvir (B), or paritaprevir + ombitasvir + dasabuvir (C,E). Posttreatment samples were obtained at the given month following end of treatment: patient A, 0 months; patient B, 4.8 months; patient C, 4.6 months; patient D, 3.1 months; and patient E, information not available. Bar graphs to the left in each panel show NS3P haplotype frequencies determined by NGS and substitution linkage analysis before treatment ( pre ) and after treatment ( post ). Haplotypes constituting more than 2% of the viral population are included in the bars; haplotypes constituting more than 20% of the viral population are highlighted in the bars. Patient B had the NS3P RAS Q80K in all haplotypes detected before and after treatment. In (A‐C), original refers to the pretreatment consensus sequence. In (D), original refers to the consensus sequence without A156V. The three line graphs to the right in (A‐C) show ORF‐wide NGS, revealing SNP frequencies along the ORF. Pre , SNP frequencies in pretreatment sequences mapped against their consensus sequence (known RASs occurring in more than 0.5% of reads are specified); Post‐all , SNP frequencies in posttreatment sequences mapped to the pretreatment consensus sequence (known RASs occurring in greater than 20% of reads are specified); Post de novo selection , frequencies of de novo selected SNPs, found in less than 0.5% of reads in pretretament sequences, mapped to the pretreatment consensus sequence (all substitutions occurring in greater than 20% of reads are specified). Known RASs are indicated in bold for NS3P using relative NS3P numbers (brown), for NS5A using relative NS5A numbers (purple), and for NS5B using relative NS5B numbers (orange). Other de novo SNPs outside the drug targets are in regular font (black), with numbers relating to the polyprotein of the H77 reference strain (GenBank identifier AF009606).

Article Snippet: Percentage of HCV infected cells was determined by immunostaining with anti‐NS5A‐9E10 and anti‐Core‐C7‐50 (Enzo Life Sciences and Abcam).

Techniques: Infection, Sequencing, Selection