Journal: bioRxiv

Article Title: Neuronal cell line expressing full-length mutant huntingtin exhibits alterations in proteolysis

doi: 10.64898/2026.01.15.699723

Figure Lengend Snippet: Application of the Sleeping Beauty system for the transposition of normal/mutant huntingtin genes into the genome of Neuro-2a cells allows to create HD transgenic cellular model and determine the levels of HttQ15/Q138 genes expression in obtained cell lines. a) The validation of transgenes expression by quantitative PCR (qPCR). SB::Q15/Q138 – pSB tet -Neo vector containing HttQ15 or HttQ138 gene, SB::(-) – pSB tet -Neo vector without huntingtin gene insertion, -SB – Neuro-2a cells without transfection, RT – reverse transcription; b) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression; c) Western blotting of poly/monoclonal transgenic Neuro-2a cells lysates without/with doxycycline induction of HttQ15 or HttQ138 expression. Data for control Neuro-2a lines (SB::(-), with transfection of «empty» vector, and -SB, without transfection, are represented also. Top panel – lysates of four different monoclonal transgenic Neuro-2a lines with HttQ15 expression, middle panel – same as top, but for lysates of HttQ138 monoclonal lines, bottom panel – lysates of polyclonal transgenic Neuro-2a lines; d) The transgene (HttQ15/HttQ138) copy number in the genomes of monoclonal (Q15m/Q138m) and polyclonal (Q15p/Q138p) Neuro-2a lines. The GAPDH-normalized Htt gene copy number in the SB::(-) control line was referred to 1. e) 2 -ΔΔCt values for transgenic Neuro-2a lines with HttQ15 or HttQ138 expression, (f) Western blotting of lysates without/with doxycycline induction of HttQ15 or HttQ138 expression after 3 years of storage, cultivation and passaging. Total protein content in each sample was controlled by β-actin immunoreactivity. Data in Tables b) and f) and Fig. d) are represented as mean ± S.D. (n = 3).

Article Snippet: The original pSB tet -Neo vector was from Addgene (#60509).

Techniques: Mutagenesis, Transgenic Assay, Expressing, Biomarker Discovery, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Reverse Transcription, Western Blot, Control, Passaging

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: Sequencing results of the inserts in CD147-TT-pSICHECK2 plasmid. Arrows indicate the inserted rs6757 TT genotype fragment

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Sequencing, Plasmid Preparation

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: The Blast of the inserts in CD147-TT-pSICHECK2 plasmid. The sequencing results were analyzed by Blast, the inserted sequence of target gene was 100% consistent with the sequence of CD147 rs6757 TT genotype in the NCBI database

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Plasmid Preparation, Sequencing

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: Sequencing results of the inserts in CD147-CC-pSICHECK2 plasmid. The sequencing result showed that the mutation from nucleotide T to C at the target site (marked in green) of CD147 gene had successfully achieved

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Sequencing, Plasmid Preparation, Mutagenesis

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: The Blast of the inserts in CD147-CC-pSICHECK2 plasmid. After mutating from nucleotide T to C, the inserted sequence was 99% consistent with the sequence of CD147 in the NCBI database by Blast analysis

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Plasmid Preparation, Sequencing

Journal: World Journal of Surgical Oncology

Article Title: Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population

doi: 10.1186/s12957-022-02724-w

Figure Lengend Snippet: The R/F analysis of a luciferase reporter vector. A Luciferase expression was significantly reduced following transfection with CD147-TT-pSICHECK2 ( P < 0.01); the translational suppression of miR-3976 can be reversed by its inhibitor. B There was no significant statistical difference between the groups following transfection with CD147-CC-pSICHECK2. C Luciferase activity was decreased by miR-3976 in dose-dependent manner for the constructs with a TT genotype but not changed for constructs with a CC genotype at the rs6757:T>C polymorphism. Each transfection was carried out in triplicate. * denotes a p value < 0.05; ** denotes a p value < 0.01

Article Snippet: The rs6757 TT genotype inserts were digested by XhoI/NotI , gel-purified, extracted, and then subcloned into the XhoI/NotI site of pSICHECK2 vector (Promega).

Techniques: Luciferase, Plasmid Preparation, Expressing, Transfection, Activity Assay, Construct

Journal:

Article Title: Neuroendocrine differentiation factor, IA-1, is a transcriptional repressor and contains a specific DNA-binding domain: identification of consensus IA-1 binding sequence

doi:

Figure Lengend Snippet: IA-1 zinc-fingers 2 and 3 are essential for transcriptional activity. Various zinc-finger constructs were fused in frame with hEgr-1 AD (a.a. 1–147). Co-transfection of zinc-finger mutants with the IA-1 –426/+40 bp promoter/pCAT3 reporter gene into β-TC-1 cells revealed that zinc-fingers 2 and 3 are the key motifs that contribute to the transcriptional activity. Zinc-finger 3 alone exhibits 50% of the control activity, whereas the combination of zinc-finger 3 and 4 only exhibits 25% of the control activity. The data are expressed as fold increase over the empty pcDNA3 expression vector. A CMV–βgal vector is used to normalize transfection efficiency. The graph represents the average of three separate experiments and SEM.

Article Snippet: The –426/+40 bp IA-1 promoter/CAT construct was generated by subcloning a Nhe I/ Xho I –426/+40 bp IA-1 promoter fragment into the Nhe I/ Xho I site of the pCAT3 vector (Promega).

Techniques: Zinc-Fingers, Activity Assay, Construct, Cotransfection, Expressing, Plasmid Preparation, Transfection