vector pgl3  (Addgene inc)

Journal: PLOS One

Article Title: Zinc finger protein 184 prevents α-synuclein preformed fibril-mediated neurodegeneration through the interleukin enhancer binding factor 3-microRNA-7 pathway

doi: 10.1371/journal.pone.0323279

Figure Lengend Snippet: (a) RT-qPCR of ILF3 mRNA levels in SH-SY5Y cells overexpressing GFP-ZNF184. (b) Immunoblot analysis of ILF3 protein levels in the GFP-ZNF184-overexpressing SH-SY5Y cells. ZNF184 antibody detects both endogenous ZNF184 (indicated by a single red asterisk) and exogenous GFP-tagged ZNF184 (indicated by two red asterisks). Quantification of ILF3 protein levels is normalized to β-actin. (c) Immunoblotting of ILF3 and ZNF184 in ZNF184 knockdown SH-SY5Y cells by siRNA-ZNF184. Quantification of the immunoblot normalized to β-actin. (d) Prediction of ZNF184 consensus sequence by expanding linear SVM. (e) ChIP assay of Flag-tagged ZNF184-overexpressing SH-SY5Y cells. Rabbit IgG and histone antibodies are used as negative and positive controls, respectively. (f) Localization of ZNF184 ChIP primer binding site in the human ILF3 promoter with ZNF184 consensus sequence, human ILF3 promoter (WT) sequence, MT1 (mutant 1), MT2 (mutant 2), and MT1/2 (mutant 1 and 2) sequences. (g) Luciferase assay of overexpressing SH-SY5Y cells with pGL3-basic vector, pGL3-hILF3 promoter (WT), mutants (MT1, MT2, and MT1/2) and Flag- ZNF184. (h, i) Immunoblotting of ILF3 in CTX (h) and SN (i) samples of a human patient with PD ( n = 3/group). Statistical significance was assessed through a one-way ANOVA and unpaired t- test, with significance levels defined as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: SH-SY5Y cells were transfected for 48 h using the pGL3-basic vector (Addgene, Watertown, MA, USA), pGL3-human ILF3 promoter (pGL3-hILF3), Flag-ZNF184, pGL3-MT1, pGL3-MT2, and pGL3-MT1/2 in Opti-MEM (Gibco, Grand Island, NY, USA) with the Xtreme-gene (Roche Holding AG, Basel, Switzerland).

Techniques: Quantitative RT-PCR, Western Blot, Knockdown, Sequencing, Binding Assay, Mutagenesis, Luciferase, Plasmid Preparation

Journal: The Journal of Clinical Investigation

Article Title: Epigenetic alteration of smooth muscle cells regulates endothelin-dependent blood pressure and hypertensive arterial remodeling

doi: 10.1172/JCI186146

Figure Lengend Snippet: ( A ) Schematic of the human JMJD3 gene, including gene structure, H3K27Ac, DHS, BP-associated SNPs, cloned fragments for luciferase assays, and vertebrate conservation (UCSC genome browser). ( B ) Luciferase results of DHS1 rs62059712 (T-major vs. C-minor) and ( C ) DHS2 rs74480102 (G-major vs. A-minor) in cultured primary human bronchial smooth muscle cells (HuBrSMCs). ( D ) qPCR for JMJD3 in HuSMCs with CRISPR/Cas9-mediated deletion of a 450 bp region encompassing rs62059712 within DHS1 (ΔDHS1) compared with unedited (WT) HuSMCs. ( E ) Sequence of rs62059712 T-major and C-minor sequences with SP1 consensus binding site underlined. ( F ) Western blot for SP1 after affinity purification using T versus C probes corresponding to rs62059712 SNP region incubated with human aortic smooth muscle cell (HuAoSMC) nuclear lysate. ( G ) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs compared with IgG negative control. ( H ) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs serum starved or treated with TGF-β. ( I ) Luciferase assays in mAoSMCs treated with siNTC and siSp1 siRNAs, then transfected with pGL3-DHS1-T and -C. ( J ) Luciferase assays in HuSMCs transfected with DHS1-C construct and then treated with TGF-β (20 ng/ml). ( K ) qPCR for Jmjd3 after NTC versus Sp1 knockdown in mAoSMCs serum starved for 16 hours. Data are represented as mean ± SEM. Results are representative of data from SMCs from 4–6 mice per group. n = 3 independent experiments. Two-tailed Student’s t test, * P < 0.05; ** P < 0.01.

Article Snippet: DNA fragments were then cloned into pGL3 luciferase basic vector (Addgene), and the correct sequence was verified by Sanger sequencing.

Techniques: Clone Assay, Luciferase, Cell Culture, CRISPR, Sequencing, Binding Assay, Western Blot, Affinity Purification, Incubation, ChIP-qPCR, Negative Control, Transfection, Construct, Knockdown, Two Tailed Test

Journal: Animals : an Open Access Journal from MDPI

Article Title: Pleiotropic Gene HMGA2 Regulates Myoblast Proliferation and Affects Body Size of Sheep

doi: 10.3390/ani14182721

Figure Lengend Snippet: Identification of HMGA2 core promoter and variation. ( A ) Relative luciferase activities of different lengths of promoter fragments; ( B ) Sanger sequencing detected SNP rs428001129 in NC_056056.1: 154134300-154134882 (-618 to -1200 bp upstream of the HMGA2 TSS) containing a core promoter; ( C ) rs428001129 changed HMGA2 promoter activity. P1, NC_056056.1: 154133741-154135682 (upstream of TSS, -59 to -2000 bp); P2, NC_056056.1: 154133741-154135281 (upstream of TSS, -59 to -1599 bp); P3, NC_056056.1: 154133741-154134882 (upstream of TSS, -59 to -1200 bp); P4, NC_056056.1: 154133741-154134300 (upstream of TSS, -59 to -618 bp). ** p < 0.01.

Article Snippet: In this study, a series of promoter deletion fragments of HMGA2 were cloned into the promoter region of the firefly luciferase gene of the pGL3-Basic vector (Addgene #212936) with the Renilla luciferase of the pRL-TK vector as an internal control.

Techniques: Luciferase, Sequencing, Activity Assay

Journal: medRxiv

Article Title: Epigenetic Alteration of Smooth Muscle Cells Regulates Endothelin-Dependent Blood Pressure and Hypertensive Arterial Remodeling

doi: 10.1101/2024.07.09.24310178

Figure Lengend Snippet: A) Schematic of the human JMJD3 gene including gene structure, H3K27Ac, DHS, blood pressure-associated SNPs, cloned fragments for luciferase assays, and vertebrate sequence conservation obtained from UCSC genome browser. Features were used to prioritize SNPs in transcriptionally relevant regions. B) Luciferase results of the DHS1 containing rs62059712 (T-major vs. C-minor) and C) DHS2 containing rs74480102 (G-major vs. A-minor) in cultured primary human smooth muscle cells (HuSMCs). D) qPCR for JMJD3 expression in HuSMCs with CRISPR-Cas9-mediated deletion of a 450 bp region encompassing the rs62059712 within DHS1 (ΔDHS1) compared with unedited (WT) HuSMCs. E) Sequence of the rs62059712 T-major and C-minor sequences with SP1 consensus binding site underlined. F) Western blot for SP1 after affinity purification using T vs. C probes corresponding to rs62059712 SNP region incubated with human aortic SMC (HuAoSMC) nuclear lysate. G) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs compared to IgG negative control. H) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs serum starved or treated with TGFβ. I) Luciferase assays in mAoSMCs treated with siNTC and siSp1 siRNAs then transfected with pGL3-DHS1-T and -C constructs. J) Luciferase assays in HuSMCs transfected with DHS1-C construct and then treated with TGFβ (20 ng/ml). K) qPCR for Jmjd3 expression after NTC vs. Sp1 knockdown in mAoSMCs. Data are presented as the mean ± SEM. Results are representative of data from SMCs from 4-6 mice per group, n=3 independent experiments. Data were first analyzed for normal distribution, and if data passed the normality test, a two-tailed Student’s t test was used, *p<0.05, **p<0.01.

Article Snippet: DNA fragments were then cloned into pGL3 luciferarase basic vector (Addgene), and the correct sequence was verified by Sanger sequencing.

Techniques: Clone Assay, Luciferase, Sequencing, Cell Culture, Expressing, CRISPR, Binding Assay, Western Blot, Affinity Purification, Incubation, ChIP-qPCR, Negative Control, Transfection, Construct, Knockdown, Two Tailed Test