Journal: Biology of Reproduction

Article Title: Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells †

doi: 10.1093/biolre/ioy097

Figure Lengend Snippet: DNA end-joining efficiency is decreased in DES vs. VEH MSCs. Luciferase gene reporter assay detected lesser DNA end-joining activity in DES vs. VEH MSCs following co-transfection with linearized HindIIIpGL2-control luc and CMV-β-gal plasmids. Means of combined experimental and biological replicates were compared using a two-tailed Student unpaired t-test. Lines represent the mean normalized end-joining activity ± SD. *P < 0.0001

Article Snippet: Plasmid DNA preparation Briefly, SV40- luc pGL2-control vector plasmid DNA ( Supplemental Figure S1A , Promega, Madison, WI) was used to transform DH5α Escherichia coli cells for plasmid amplification and was then isolated using PureYield Plasmid Midiprep System (Promega).

Techniques: Luciferase, Reporter Assay, Activity Assay, Cotransfection, Two Tailed Test

Journal:

Article Title: The Antiapoptotic Gene mcl-1 Is Up-Regulated by the Phosphatidylinositol 3-Kinase/Akt Signaling Pathway through a Transcription Factor Complex Containing CREB

doi:

Figure Lengend Snippet: (A) CRE-2 and SIE binding motifs can individually confer IL-3 inducibility on a heterologous promoter (SV40 minimal promoter) and can work additively in IL-3 stimulation of the same reporter system. Ba/F3 cells transfected with various reporter plasmids as indicated were left untreated (−IL-3) or stimulated with IL-3 (+IL-3) before cell lysates were prepared and analyzed for luciferase activity. Data shown here are representative of three independent experiments performed in triplicate. (B) IL-3 stimulation of the CRE-2 but not the SIE reporter is mediated through the PI3-K/Akt kinase pathway. Ba/F3 cells transfected with the reporter plasmids as indicated plus an expression vector encoding nothing (Vector), DNAkt, or DNp85 were processed and analyzed for the luciferase activities as described in the legend to Fig. ​Fig.7C.7C. The data shown here are averages from five to six independent experiments, and the reporter activities of cells transfected with each reporter but treated under various conditions are plotted relative to cells transfected by the same reporter plasmid and deprived of IL-3. The latter activity was considered to be 100. The IL-3 induction of each reporter was statistically significant compared to that of the parental reporter (pGL2 promoter), as was the dominant negative effect of DNp85 or DNAkt on the CRE-2 or the SC reporter. The P values are as follows: ∗, 0.01 < P < 0.02; ∗∗, 0.001 < P < 0.01; ∗∗∗, P < 0.001; #, 0.02 < P < 0.05.

Article Snippet: Plasmids pGL2-1XCRE-2, pGL2-1XSIE, and pGL2-1XSC were derived by inserting one copy of the DNA fragment containing CRE-2, SIE, or both sites (see below for individual sequences) into the Sma I site of the pGL2-promoter vector (Promega).

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Dominant Negative Mutation

Journal: Oncotarget

Article Title: Sp1-mediated microRNA-182 expression regulates lung cancer progression

doi:

Figure Lengend Snippet: (A) Scramble (shScr) and different doses of Sp1 shRNAs (shSp1) were transfected into A549 for 48 h. The miR-182 level was determined by stem-loop RT-PCR. U6 served as the internal control (panel a). Data were quantified after three independent experiments (panel b). (B) Different titer of adeno-GFP-Sp1 virus was infected IMR-90 cells for 48 h. The miR-182 level was determined by stem-loop RT-PCR (panel a). Data were quantified after three independent experiments (panel b). (C) Plasmids, pGL2 or pGL2-miR-182 (-1000/+50), and GFP or GFP-Sp1 were co-transfected into H1299 cells for 24h. Cells were harvested to study the luciferase activity. Data were quantified after three independent experiments. (D) The plasmids, pGL2 or pGL2-miR-182, were transfected into H1299 cells with mithramycin A treatment for 24 h. Cells were harvested for luciferase activity assays. (E) Schematic diagram indicates the location of putative Sp1 binding sites on miR-182 promoter region (panel a). ChIP assays were performed with anti-acetyl-H3 (panel b), and anti-Sp1 antibodies (panel c). DNA was extracted for PCR with miR-182 and p21 primers. Data were quantified after three independent experiments (panel d). (F) A549 cells were harvested for DAPA with a biotin-conjugated p21 and miR-182 promoter probes, and samples were analyzed by Western blotting using anti-Sp1 antibodies (panel a). Data were quantified after three independent experiments (panel b). (G) Plasmids, GFP or GFP-Sp1, were co-transfected with pGL2, pGL2-miR-182 WT or mutation plasmids into H1299 cells for 24 h, and then cells were harvested for luciferase activity assays. Data are representative of three independent experiments, each of which was performed in triplicate, and presented as the mean ± SEM. The level of statistical significance determined by t -test (*, p<0.05; **, p<0.01).

Article Snippet: After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI and BglII for pGL2-miR-182, MluI and BglII for pGL2-FOXO3 (New England Biolabs, Ipswich, MA).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Infection, Luciferase, Activity Assay, Binding Assay, Western Blot, Mutagenesis

Journal: Oncotarget

Article Title: Sp1-mediated microRNA-182 expression regulates lung cancer progression

doi:

Figure Lengend Snippet: Total RNA and cell lysates were prepared from indicated cell lines (A) or from clinical lung tissues of lung cancer patients (B). The miR-182 level was determined by stem-loop RT-PCR and Sp1 level was studied by Western blotting with anti-Sp1 antibodies. U6 and tubulin served as the internal control. (C) Total RNA and cell lysates were prepared from 32 paired normal lung tissues and lung adenocarcinoma samples. The miR-182 level was studied by stem-loop RT-PCR and Sp1 levels were studied by RT-PCR. (D) The relationship between Sp1 and the miR-182 level in the 32 lung cancer samples was statistically analyzed using Fisher's exact test.

Article Snippet: After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI and BglII for pGL2-miR-182, MluI and BglII for pGL2-FOXO3 (New England Biolabs, Ipswich, MA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: Sp1-mediated microRNA-182 expression regulates lung cancer progression

doi:

Figure Lengend Snippet: (A) Lenti-miRZip and lenti-miRZip-182 viruses were infected into H1299 for 96 h individually. FOXO3 level was studied by Western blotting with anti-FOXO3 antibodies and miR-182 level was studied by stem-loop RT-PCR. (B) Plasmids, pGL3 and pGL3-FOXO3-3'UTR, were transfected into miRZip and miRZip-182 stably expressed H1299 cells for 24 h, and then cells were harvested for luciferase activity assays. (C) Different doses of GFP-Sp1 adenovirus were infected into the miRZip and miRZip-182 stably expressed H1299 cells for 48 h. FOXO3 level was studied by Western blotting using anti-FOXO3 antibodies (panel a). Quantitative results from three independent experiments are shown (panel b). The level of statistical significance was determined by t -test (*, p<0.05; ***, p<0.001).

Article Snippet: After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI and BglII for pGL2-miR-182, MluI and BglII for pGL2-FOXO3 (New England Biolabs, Ipswich, MA).

Techniques: Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Stable Transfection, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Sp1-mediated microRNA-182 expression regulates lung cancer progression

doi:

Figure Lengend Snippet: (A) The Sp1 and FOXO3 levels in clinical lung tissue samples were studied by IHC staining using antibodies against Sp1 and FOXO3, respectively. (B) Cell lysates were harvested from various cell lines for Western blotting using antibodies against FOXO3 and Sp1, and tubulin as an internal control. (C) Adeno-GFP-Sp1 viruses were infected into IMR-90 cells for 48 h, and FOXO3 mRNA and protein were studied by RT-PCR and Western blotting, respectively. GAPDH served as the internal control (panel a). Scramble and Sp1 shRNAs were transfected into H1299 for 48 h, then FOXO3 mRNA and protein levels were studied by RT-PCR and Western blotting (panel b). (D) Scramble and Sp1 shRNAs were transfected into H1299 for 48 h, and then cells were harvested at indicated time points following cycloheximide treatment for studying the Sp1 and FOXO3 levels with Western blotting. The levels of FOXO3 protein from three independent experiments were quantified using tubulin as an internal control. (E) Plasmids, pGL2 or pGL2-FOXO3 (-1000/+50), were cotransfected with GFP or GFP-Sp1 into H1299 cells for 24 h, then cell lysates were harvested for luciferase activity assays. (F) Adeno-GFP-Sp1 viruses were infected into H1299 cells for 24 h, and cells were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Cells lysates were harvested for luciferase activity assays. (G) H1299 cells, which were infected with GFP-Sp1 adenovirus for 24 h, were then transfected with pGL3 or pGL3-FOXO3-3'UTR plasmid for 24 h. Total RNA was extracted at various time points following actinomycin D treatment. The mRNA levels of luciferase were determined by using quantitative RT-PCR and quantified using GAPDH as an internal control. Data are representative of three independent experiments, each of which was performed in triplicate, and presented as the mean ± SEM. The level of statistical significance determined by t -test (*, p<0.05; **, p<0.01).

Article Snippet: After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI and BglII for pGL2-miR-182, MluI and BglII for pGL2-FOXO3 (New England Biolabs, Ipswich, MA).

Techniques: Immunohistochemistry, Western Blot, Infection, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Sp1-mediated microRNA-182 expression regulates lung cancer progression

doi:

Figure Lengend Snippet: (A) Immunofluorescent staining of Alexa Fluor 568-conjugated phalloidin that is a high-affinity probe for F-actin (red) in miRZip and miRZip-182 stably expressed H1299 cells. DNA was stained with DAPI (blue). Stained cells were photographed under a fluorescence microscope at x 600 magnification. (B) Confluent monolayers of miRZip or miRZip-182 stably expressed H1299 cells were wounded and incubated for an additional 16 h (panel a). Migratory area was calculated for quantification (panel b). (C) The migration activities of H1299 cells (2 x 10 4 ) expressing miRZip or miRZip-182 were studied by Transwell chambers. (D) The miRZip or miRZip-182 stably expressed H1299 cells (4 x 10 6 ) were suspended in 100 μl of PBS and injected into the lateral tail vein of SCID mice. After 8 weeks, all mice were killed and the number of pulmonary tumor nodules was calculated after fixation of lungs with 4% formaldehyde for 48 h (panel a), and the number of pulmonary metastatic tumor nodules was counted (panel b). (E) FOXO3 and miR-182 in H1299 cells were knockdown by shFOXO3 and miRZip-182 respectively, and then migration of cells (3 x 10 4 ) was studies by Transwell chambers (panel a). In addition, cell lysates were harvested from FOXO3 and miR-182 knockdown cells for Western blotting using antibodies against N-cadherin, β-catenin, vimentin, FOXO3 and tubulin (panel b), respectively. (F) Heat map of the 19 of genes from miRZip and miRZip-182 microarray data, the red color represents genes that are upregulated and the green color represents genes that are downregulated. The level of statistical significance determined by t -test (*, p<0.05; **, p<0.01; ***, p<0.001).

Article Snippet: After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI and BglII for pGL2-miR-182, MluI and BglII for pGL2-FOXO3 (New England Biolabs, Ipswich, MA).

Techniques: Staining, Stable Transfection, Fluorescence, Microscopy, Incubation, Migration, Expressing, Injection, Western Blot, Microarray

Journal: Oncotarget

Article Title: Sp1-mediated microRNA-182 expression regulates lung cancer progression

doi:

Figure Lengend Snippet: (B) Schematic diagram illustrates Sp1 regulates miR-182 to silence FOXO3 expression in early and late stages of lung cancer progression.

Article Snippet: After amplification and purification, the DNA fragments were ligated to pGL2 vector using restriction enzymes, KpnI and BglII for pGL2-miR-182, MluI and BglII for pGL2-FOXO3 (New England Biolabs, Ipswich, MA).

Techniques: Expressing