Journal: Molecular Cancer Therapeutics

Article Title: Antileukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.mct-18-0706

Figure Lengend Snippet: Figure 5. Notch-mediated PLK1 ubiquitination involves PARylation of CHFR1 (checkpoint ubiquitin E3 ligase) in B-ALL. A, PLK1 mRNA expression was determined by qRT- PCR after coculture with HS5-DLL1 or HS5-GFP for 48 hours (n ¼ 3). B, B-ALL cell lines were treated with MG132 4 hours prior to various time points (12, 24, or 48 hours) of coculture with HS5-GFP or HS5-DLL1. Cell lysates were subjected to immunoprecipitation with ubiquitin and probed for PLK1. C, Similarly, after 48 hours of coculture, cells were treated with MG132 for 4 hours and harvested. Cell lysates were subjected to immunoprecipitation with PLK1, and blots were probed with PLK1 and ubiquitin. D, The same panel of B-ALL cells was transduced with retrovirus-mediated HES1 for 48 hours to induce ectopic expression of the gene. The cells were treated with MG132 and the cell lysates subjected to immunoprecipitation with ubiquitin and probed for PLK1. E, Primary B-ALL cells (Pt. B1) were either transduced with HES1 retrovirus, transfected with CHFR siRNA (siCHFR), or both for 24 hours; these cells and untreated controls were probed for CHFR and PLK1 expression. F, Cell lysates from B-ALL cell lines coculture with HS5-GFP or HS5-DLL1 were immunoprecipitated with CHFR and probed for PLK1. G, T-ALL cells (CEM) and B-ALL cells (SB, JM1, Nalm6) were either cocultured on control or DLL1-expressing HS5 cells and subjected to CHFR depletion via siRNA for 4 days. Viability was determined by Trypan blue. H, Cell lysates from E were immunoprecipitated with p53, and blots were probed with pMDM2(ser260) and p53. I, B-ALL cells were cocultured on control or DLL1-expressing HS5 cells for 48 hours, and cell lysates were immunoprecipitated with CHFR and resolved on 4% to 15% non-denaturing gradient gels; the membrane was probed for polyADP ribosylation (PAR). J, Cells from patients with Ph-like B-ALL (Pt.B1, Pt.B2) were cocultured on control or DLL1-expressing HS5 cells and treated for 48 hours with the PARP inhibitor, 3ABA. Viability was determined by Trypan blue (, P < 0.05; , P < 0.01).

Article Snippet: Antibodies b-actin (A2066; Sigma-Aldrich), cleaved Notch2 (C651.6DbHN; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), HES1 (ab71559; Abcam; TA500014; Origene), Notch-1, -3, and -4 (ab52627, ab23426, and ab184742; Abcam), Jagged1 (sc11376; Santa Cruz Biotechnology), DLL1 (sc73899; Santa Cruz Biotechnology), p53 (DO1; Santa Cruz Biotechnology), FITC anti-p53 antibody (645803; BioLegend), PLK1 (ab17057; Abcam), CHFR (H00055743M01; Abnova), PAR (4335-MC-100-AC; Trevigen), MDM2 (OP115; Millipore), pMDM2(ser260) (orb129684; Biorbyt LLC; Bioworld Technology, Inc.), phycoerythrin-labeled donkey antirabbit IgG antibody (406421; BioLegend), and anti-ubiquitin (U5379; Sigma-Aldrich) were used.

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Immunoprecipitation, Transduction, Transfection, Control, Membrane

Journal: Molecular Cancer Therapeutics

Article Title: Antileukemia Effects of Notch-Mediated Inhibition of Oncogenic PLK1 in B-Cell Acute Lymphoblastic Leukemia

doi: 10.1158/1535-7163.mct-18-0706

Figure Lengend Snippet: Figure 6. Pharmacologic inhibition of PLK1 shows antileukemia activity in Ph-like B-ALL. A, Cells representing T-ALL (CEM) or B-ALL (SB, JM1, Nalm6 [N6]) and primary samples from patients with Ph-like B-ALL were treated with a vehicle control or PLK1 inhibitor, BI2536 (100 nmol/L), for 1 week. Cells were counted by Trypan blue daily and the counts normalized the seed number. B and C, Intracellular p53 (B) and pMDM2 (ser260; C) for all cells in A were determined by flow cytometry at 48 hours. D, Similarly, cell lysates were collected at the same time point and probed for pMDM2 (ser260), MDM2, p53, and b-actin. E, NSG-SGM3 mice engrafted with cells from a patient with Ph-like B-ALL (Pt.B1) were treated, beginning 3 weeks after initial tail vein injection, with vehicle (control), PLK1 inhibitor BI2536 (15 mg/kg), or BI6727 (volasertib; 15 mg/kg) by gavage twice per week for 2 weeks (n ¼ 10 mice per group) and continuously monitored for 2 additional weeks. The leukemia burden was measured weekly in the peripheral blood by hCD45 staining. F, Mice were euthanized 4 weeks after beginning treatment, and the leukemia burden was measured in the bone marrow and spleen by hCD45 staining. G, Intracellular pMDM2 and p53 levels in the bone marrow of the treated mice were quantified by flow-based analysis. H, SB B-ALL was treated with various combinations of dexamethasone (DEX) or vincristine (Vi) and PLK1 inhibitor BI2536 (BI) for 3 days (n ¼ 3 per treatment group). Cell viability was determined by Alamar blue (, P < 0.05; , P < 0.01).

Article Snippet: Antibodies b-actin (A2066; Sigma-Aldrich), cleaved Notch2 (C651.6DbHN; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), HES1 (ab71559; Abcam; TA500014; Origene), Notch-1, -3, and -4 (ab52627, ab23426, and ab184742; Abcam), Jagged1 (sc11376; Santa Cruz Biotechnology), DLL1 (sc73899; Santa Cruz Biotechnology), p53 (DO1; Santa Cruz Biotechnology), FITC anti-p53 antibody (645803; BioLegend), PLK1 (ab17057; Abcam), CHFR (H00055743M01; Abnova), PAR (4335-MC-100-AC; Trevigen), MDM2 (OP115; Millipore), pMDM2(ser260) (orb129684; Biorbyt LLC; Bioworld Technology, Inc.), phycoerythrin-labeled donkey antirabbit IgG antibody (406421; BioLegend), and anti-ubiquitin (U5379; Sigma-Aldrich) were used.

Techniques: Inhibition, Activity Assay, Control, Cytometry, Injection, Staining

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: bioRxiv

Article Title: Novel Quinazoline derivatives inhibited HCV Serine protease and viral replication in Huh-7 cells

doi: 10.1101/671313

Figure Lengend Snippet: The cells were then treated with increasing concentrations of compound 4 for 72 h. The fixed cells were treated with an antibody specific to the HCV NS3 protein labeled with FITC fluorescence dye. The results showed that the inhibitory effect of compound 4 towards HCV replicon replication is dose-dependent.

Article Snippet: A mouse antibody specific to the HCV NS3 protein labeled with FITC fluorescence dye (biorbyt, UK, Cat. # orb15740) was added, and the cells were incubated for 3h at 4°C.

Techniques: Labeling, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Deficiency in Kelch protein Klhl31 causes congenital myopathy in mice

doi: 10.1172/JCI93445

Figure Lengend Snippet: (A) Coimmunoprecipitation assay showing Klhl31 interacts with FlnC. (B) Transfected COS-7 cells were immunoprecipitated with anti-Flag antibody, followed by Western blotting with anti-HA antibodies. Polyubiquitination of FlnC is reduced in the presence of a K48R mutant Ub or in the absence of Klhl31. FlnC protein levels were reduced by overexpression of Klhl31, Cul3, and Ub. This reduction is rescued by the addition of the K48R mutant Ub or in the absence of Klhl31. Black arrowhead indicates increased ubiquitinated FlnC with a reduction in polyubiquitination in the presence of the K48R mutant Ub. White arrowhead indicates a nonspecific band. (C) Immunohistochemistry of WT and Klhl31-KO quadriceps with α-actinin and FlnC antibodies shows FlnC aggregates in Klhl31-KO mice. Scale bar: 20 μm. (D) Western blot analysis of WT and Klhl31-KO quadriceps at P10 using FlnC, Slmap, and Usmg5 antibodies.

Article Snippet: Membranes were blocked for 1 hour at room temperature with 5% nonfat dry milk in TBST, and primary antibody hybridization was carried out overnight at 4˚C using the following antibodies: Klhl31, 1:100 (sc-132316, Santa Cruz Biotechnology Inc.); Ckap4, 1:1000 (A302-257A-T, Bethyl); Slmap, 1:1000 (A304-505A-T, Bethyl); Usmg5, 1:2000 (17716-1-AP, Proteintech); MybpC2, 1:1000 (SAB2108180, Sigma-Aldrich); FlnC, 1:1000 (ab180941, Abcam); Gapdh, 1:10,000 (MAB374, Millipore); Myc 1:1000 (51-1485GR, BD Biosciences); Flag, 1:1000 (F3165, Sigma-Aldrich); Flag-HRP, 1:2000 (A8592, Sigma-Aldrich), and Tubulin, 1:5000 (T6199, Sigma-Aldrich).

Techniques: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Mutagenesis, Over Expression, Immunohistochemistry

Journal: PLoS ONE

Article Title: Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot

doi: 10.1371/journal.pone.0146710

Figure Lengend Snippet: (A) The proposed secondary structure of HCV IRES. The IRES region spanning nts 277–343 (shown in gray) was targeted by siRNAs. The target site of the selected potent anti-HCV siRNA siIE22 is shown in blue. The base-pairings in the proposed pseudoknot (PK) structures (PKs 1 and 2) are shown in green. (B) siRNA sequences tiled over HCV IRES. The underlined sequence represents a mapped druggable region (nts 313–343) where the targets of selected potent siRNAs were enriched. (C) Anti-HCV activity of HCV IRES-targeting siRNAs in Huh7 cells transfected with Rluc-JFH1 (top panel), an HCV replicon encoding the Rluc reporter. The Rluc gene was fused in frame to the DNA sequence encoding 17 N-terminal amino acid residues of the HCV core protein. Huh7 cells were electroporated with the Rluc-JFH1 in vitro RNA transcript and pGL3 plasmid used for normalization of transfection efficiency. After 24 h, the cells were transfected with each of the IRES-specific siRNAs or a scrambled (Sc) siRNA (50 nM). At 48 h post-transfection, luciferase activity was measured. (D) Huh7 cells harboring an HCV subgenomic replicon RNA (R-1) were transfected with each of the IRES-specific siRNAs or Sc siRNA (10 nM each). At 48 h post-transfection, HCV RNA levels were quantified by real-time qRT-PCR. *, P < 0.01. (E and F) Dose-dependent inhibition of HCV-replication by siIE22 was assessed in R-1 cells, as described in (D). HCV genome copy number and HCV proteins (NS5A and NS5B) levels were analyzed by qRT-PCR (E) and western blotting (F), respectively.

Article Snippet: The goat anti-HCV E2 polyclonal antibody was obtained from Acris Antibodies (GmbH, Hideenhausen, Germany).

Techniques: Sequencing, Activity Assay, Transfection, In Vitro, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Inhibition, Western Blot

Journal: PLoS ONE

Article Title: Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot

doi: 10.1371/journal.pone.0146710

Figure Lengend Snippet: (A) Passenger strand sequences of siIE22 and six siRNAs sharing their targets with siIE22 in the IRES subdomain IIIf. (B) Evaluation of anti-HCV efficacy of a set of selected siRNAs (100 pM each) using an HCV replicon expressing an Rluc reporter. Luciferase activity at 48 h post-siRNA treatment is shown. Sc, scrambled siRNA. (C) Antiviral efficacy of gs_PS1 siIE22 and siIE318_27 in R-1 cells harboring an HCV subgenomic replicon. (D) In vitro target cleavage assays were performed as described in . P, 5′ 32 P-radiolabeled 31-nt long HCV IRES probe; CP, cleaved probe. (E) Comparison of antiviral activity of indicated siRNAs targeting the region shared with siIE22 in R-1 cells. In (B), (C), and (E), *, P < 0.05; **, P < 0.01.

Article Snippet: The goat anti-HCV E2 polyclonal antibody was obtained from Acris Antibodies (GmbH, Hideenhausen, Germany).

Techniques: Expressing, Luciferase, Activity Assay, In Vitro, Comparison

Journal: PLoS ONE

Article Title: Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot

doi: 10.1371/journal.pone.0146710

Figure Lengend Snippet: (A) Non-modified siIE22 (2 μM) was incubated in 45% human plasma for the indicated time periods. RNA extracted from each sample was resolved by electrophoresis on a denaturing 15% polyacrylamide gel and subjected to northern blotting (NB) analysis for detection of siIE22 guide-strand. The Phosphorimager image shown is from one representative experiment of three independent experiments with similar results. Densitometric analysis of siIE22 guide strand signal was done using a Phosphorimager. Relative intensity of signals was plotted using SigmaPlot to estimate siIE22 guide strand half-life. Relative signal (% of signal at time 0) is shown below a representative blot. (B) Sequences of modified guide and passenger strands of siIE22 derivatives used in this study. Modified residues are shown in green or blue. “s”, phosphorothioate linkage. (C) Plasma stability of a set of the selected siIE22 derivatives was evaluated as in (A). (D) Anti-HCV activity of the selected modified siIE22 (1 nM) was evaluated in R-1 cells as in . *, P < 0.01. (E) Analysis of half-life of the gs_PS1 siIE22 as in (A).

Article Snippet: The goat anti-HCV E2 polyclonal antibody was obtained from Acris Antibodies (GmbH, Hideenhausen, Germany).

Techniques: Modification, Incubation, Clinical Proteomics, Electrophoresis, Northern Blot, Activity Assay

Journal: PLoS ONE

Article Title: Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot

doi: 10.1371/journal.pone.0146710

Figure Lengend Snippet: (A) HCV genome in the HCV-replicating Huh7 (+ HCV) or Huh7 (- HCV) sc xenograft was detected by northern blotting. An in vitro transcribed HCV RNA genome was used as a size marker. The 28S rRNA detected by ethidium bromide staining is shown as a loading control. (B) Immunostaining for HCV viral proteins (E2 and NS5B) in the xenograft at 4 weeks post-xenografting. DAPI, nuclear staining. Scale bar, 10 μm. (C) The NOD-SCID mice (n = 3) carrying HCV-replicating Huh7 xenograft were treated with gs_PS1 siIE22 LNP at a dose of 1 mg/kg body weight via tail vein injection. Shown are relative serum HCV RNA titers at the indicated time points. *, P < 0.01. (D) Relative serum HCV RNA titers in the mice treated with LNP-formulated Sc siRNA (Sc LNP) or gs_PS1 siIE22 LNP (1 mg/kg) once in every three days for 4 times. The data were generated from two independent experiments with a total of 6 mice per group. Each differently colored diamond represents an individual mouse. *, P < 0.01.

Article Snippet: The goat anti-HCV E2 polyclonal antibody was obtained from Acris Antibodies (GmbH, Hideenhausen, Germany).

Techniques: Northern Blot, In Vitro, Marker, Staining, Control, Immunostaining, Injection, Generated