Journal: PLoS ONE

Article Title: Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

doi: 10.1371/journal.pone.0260144

Figure Lengend Snippet: A GHRH (1–44) expression of bacterial colonies expressing His- and His-GHRH vectors demonstrated by immunoblotting. 1 E . coli HB101 His vector clone, 2 E . coli HB101 His-GHRH vector clone. B Purified His tagged bacterial GHRH 1–44 ligand was demonstrated by immunoblotting via anti-GHRH and anti-His tag antibodies, respectively. 1 His-GHRH expressing total bacterial lysate, 2 His-GHRH expressing solubilized pellets. Eukaryotic GHRH NH2 (1–44) expression in His-GHRH vector-transfected HEK293 cells was identified by C qRT-PCR, D immunoblotting, and E immunofluorescence. 1 HEK293 wt 2 HEK293 His-transfected 3 HEK293 His-GHRH transfected. F GHRH 1–44 concentration in the media of HEK293 cells was measured by Human GHRH ELISA. GAPDH was used as a loading control. 18S was used as an internal control. DAPI was used to indicate the nuclei of the cells.

Article Snippet: 100 μL of concentrated cellular media were used for GHRH ELISA assay (Elabscience, E-EL-H1146) according to the manufacturer’s instructions and were measured at 450 nm wavelength by a microplate reader (Multiskan, Thermo Scientific).

Techniques: Expressing, Western Blot, Plasmid Preparation, Purification, Transfection, Quantitative RT-PCR, Immunofluorescence, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

doi: 10.1371/journal.pone.0260144

Figure Lengend Snippet: A First bead based aptamer selection against GHRH NH2 (1–44) target and B second bead based aptamer selection against GHRH NH2 (1–29) target was performed by using the X-aptamer kit. Tube #1: Cleaved oligunucleotide pool, Tube #2: Cleaved oligonucleotide pool+Magnetic beads+Prokaryotic GHRH (1–44) target, Tube #3: Cleaved oligonucleotide pool+Magnetic beads+Eukaryotic GHRH (1–44) target, Tube #7: Cleaved oligonucleotide pool+Magnetic beads. Cycle course PCR (up) and last PCR (down) amplification of the x-aptamer selection process were given. C Sequences of putative x-aptamers were determined by next-generation sequencing. D. Analysis for all 24 putative aptamers were performed by using MEME program ( https://meme-suite.org/meme/ ).

Article Snippet: 100 μL of concentrated cellular media were used for GHRH ELISA assay (Elabscience, E-EL-H1146) according to the manufacturer’s instructions and were measured at 450 nm wavelength by a microplate reader (Multiskan, Thermo Scientific).

Techniques: Selection, Magnetic Beads, Amplification, Next-Generation Sequencing

Journal: PLoS ONE

Article Title: Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

doi: 10.1371/journal.pone.0260144

Figure Lengend Snippet: The binding affinity of putative X-aptamers was determined by dot blot assay against A GHRH NH2 (1–44) target and B GHRH NH2 (1–29) target. C Dose-dependent dot blot assay was performed in increasing doses (0–1000 nM) of X-aptamers. The scramble aptamer was used a negative control for dot-blot analysis. Dot intensities were measured by Image J (imagej.nih.gov/ij/) and analyzed by GraphPad Prism 8.0. Nonlinear regression analysis was performed by Sigma Plot v14.0 and K d was calculated. D. The specific binding affinity of selected aptamers against GHRH peptide was determined by both GHRH sandwich ELISA assay (left panel) and dot-blot analysis (right panel) in a dose-dependent manner.

Article Snippet: 100 μL of concentrated cellular media were used for GHRH ELISA assay (Elabscience, E-EL-H1146) according to the manufacturer’s instructions and were measured at 450 nm wavelength by a microplate reader (Multiskan, Thermo Scientific).

Techniques: Binding Assay, Dot Blot, Negative Control, Sandwich ELISA

Journal: PLoS ONE

Article Title: Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

doi: 10.1371/journal.pone.0260144

Figure Lengend Snippet: A GHRH expression was figured out in MIA PaCa-2 pancreatic cancer, HT-29 colorectal cancer, PC3 prostate cancer, and PNT1a normal prostate epithelium cell lines by immunofluorescence. DAPI was used to observe the nuclei of the cells. B Translational expression of GHRH in MIA PaCa-2, HT-29, PC3, and PNT1a cells was determined by immunoblotting. ß-actin was used as a loading control. C. GHRH concentrations in media of MIA PaCa-2, HT-29, PC3, and PNT1a cells determined by Human GHRH ELISA.

Article Snippet: 100 μL of concentrated cellular media were used for GHRH ELISA assay (Elabscience, E-EL-H1146) according to the manufacturer’s instructions and were measured at 450 nm wavelength by a microplate reader (Multiskan, Thermo Scientific).

Techniques: Expressing, Immunofluorescence, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

doi: 10.1371/journal.pone.0260144

Figure Lengend Snippet: The binding position of A TKY2.T1.08, C TKY2.T1.13, E TKY.T2.08, G TKY.T2.09 x-aptamers were investigated by immunofluorescence. Streptavidin-Alexa Fluor 488 conjugate was used to specify biotin-labelled x-aptamers. Cells were treated with X-aptamers in a dose-dependent manner (0–500 nM). Following incubation, cells were examined under a fluorescence microscope (Olympus) and images were taken and analyzed. The binding position of B TKY2.T1.08, D TKY2.T1.13, F TKY.T2.08, H TKY.T2.09 x-aptamers investigated by co-immunofluorescence. Streptavidin-Alexa Fluor 488 conjugate was used to specify biotin-labelled X-aptamers. Streptavidin-Alexa Fluor 588 conjugated anti-GHRH was used to specify GHRH. Cells were treated with x-aptamers in a dose-dependent manner (0–500 nM). Following incubation, cells were examined under a fluorescence microscope (Olympus) and images were taken and analyzed. GHRH siRNA was used as a negative control.

Article Snippet: 100 μL of concentrated cellular media were used for GHRH ELISA assay (Elabscience, E-EL-H1146) according to the manufacturer’s instructions and were measured at 450 nm wavelength by a microplate reader (Multiskan, Thermo Scientific).

Techniques: Binding Assay, Immunofluorescence, Incubation, Fluorescence, Microscopy, Negative Control

Journal: PLoS ONE

Article Title: Synthesis and characterization of novel ssDNA X-aptamers targeting Growth Hormone Releasing Hormone (GHRH)

doi: 10.1371/journal.pone.0260144

Figure Lengend Snippet: A. The effect of TKY2.T1.08, TKY2.T1.13, TKY.T2.08, TKY.T2.09 x-aptamers on the intracellular cAMP levels were determined by cAMP assay kit in both HT29 and MIA Paca-2 cells. B. The impact of TKY2.T1.08, TKY2.T1.13, TKY.T2.08, TKY.T2.09 x-aptamer on GH/GHRHR expression profile was investigated by co-immunofluorescence assay under a fluorescence microscope (Olympus). GHRH peptide (500 nM) was used as a positive control. Each experiment was performed and repeated at least two times, given figures are the representative figure of one of the three replicated assays. ns: non-specific, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: 100 μL of concentrated cellular media were used for GHRH ELISA assay (Elabscience, E-EL-H1146) according to the manufacturer’s instructions and were measured at 450 nm wavelength by a microplate reader (Multiskan, Thermo Scientific).

Techniques: cAMP Assay, Expressing, Immunofluorescence, Fluorescence, Microscopy, Positive Control