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: Endocrinology

Article Title: In utero exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate induces long-term changes in gene expression in the adult male adrenal gland.

doi: 10.1210/en.2013-1921

Figure Lengend Snippet: Figure 6. Serum markers that correlate with in utero exposure to DEHP. A, Heatmap of differentially expressed genes at PND21 and PND60 in offspring of rats exposed in utero to 100- or 300-mg DEHP/kgd was constructed to identify genes altered at both time points and doses. Selected gene targets (bold) were used to evaluate putative serum markers. Serum levels of AQP7 (B), FABP4 (C), and PCK1 (D) in adult rats exposed in utero to the indicated doses of DEHP. Data in C and D are means SD (n 4); **, ANOVA, P .01. E, Relationship between FABP4 and PCK1 adult rat serum levels to in utero DEHP exposure doses used. FABP4, correlation coefficient 0.37, P .49. PCK1, correlation coefficient 0.77, P .1.

Article Snippet: ELISAs were used to quantify aquaporin 7 (AQP7) (catalog number CSB-EL001967RA; Cusabio Biotech, Cedarlane Laboratories), fatty acid-binding protein 4 (FABP4) (catalog number CSB-E13027r; Cusabio Biotech), and phosphoenolpyruvate carboxykinase I (PCK1) (catalog number sE90936Ra; USCN Life Science, Inc, Cedarlane Laboratories), according to the instructions of each manufacturer.

Techniques: In Utero, Construct

Journal: Diseases

Article Title: Assessing Anti-Adipogenic Effects of Mango Leaf Tea and Mangiferin within Cultured Adipocytes

doi: 10.3390/diseases11020070

Figure Lengend Snippet: Incubating cultured adipocytes with MLT augments the secretion of adiponectin and enhances glucose uptake. ( A , B ) Adipocytes were treated with vehicle, MGF, or MLT. ELISA was used to measure secretory adiponectin ( A ), and a fluorometric kit was used for measuring glucose uptake of fluorescently labeled 2-NBDG ( B ). ( A ) Four independent experiments were conducted using one biological replicate for each experiment. ( B ) Two independent experiments were performed, each with triplicate biological replicates used. ( A , B ) Bars are group means.

Article Snippet: We measured adiponectin in the centrifuged cell culture medium using a mouse adiponectin ELISA kit (AccuSignal TM ELISA Kit, KOA0366; Rockland, Limerick, PA, USA), following the manufacturer’s instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling