Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 1. Analysis of rat AR promoter constructs in transfected rat liver cells. A, scheme of rat AR promoter con- structs used in the transfection experi- ments. B, the luciferase activity (relative luciferase activity) from rat liver cells transfected with constructs 1 to 9 and subsequently grown in isotonic medium (nonstress, open bar) or hypertonic me- dium (stress, filled bar) for 18 h. Error bars indicate standard deviation (S.D.). C, luciferase activity 6 S.D. under isotonic or hypertonic conditions. pX/p1 indicates the increase over p1 luciferase activity. Ratio H/I indicates the ratio between hy- pertonic and isotonic activity; n indicates the number of replicates.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: Construct, Transfection, Luciferase, Activity Assay, Standard Deviation

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 2. Exonuclease III-mediated DNA footprinting of promoter region necessary for osmotic response. Nuclei isolated from rat liver cells transfected with luciferase reporter construct 4 were treated with ExoIII as described under “Experimental Procedures.” Purified DNA was then subjected to linear PCR. Lane 1, linear PCR with primer 1 on HindIII-digested control template; 2, linear PCR with primer 1 on HindIII and ExoIII-digested transient template; 3, linear PCR with primer 2 on Asp718-digested control template; 4, linear PCR with primer 2 on Asp718 and ExoIII-digested transient template. The two horizontal complementary nucleotide sequences contain the primer locations and the ExoIII protected sequence is indicated by the dotted vertical lines. Sequencing reactions (A, C, G, T) were performed with the same primers used for linear PCR.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: DNA Footprinting, Isolation, Transfection, Luciferase, Construct, Purification, Control, Sequencing

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 3. Dimethyl sulfate-mediated in vivo DNA footprinting of the promoter region involved in increased constitutive activity and osmotic response. Dimethyl sulfate-mediated in vivo DNA footprinting was performed on rat liver cells cultured under normal conditions or under osmotic stress as described under “Experimental Procedures.” Lane 1, LM-PCR of DNA from cells in hyperosmotic medium for 3 h (stress 3 h/in vivo); 2, LM-PCR of DNA from cells in hyperosmotic medium for 30 min (stress 309/in vivo); 3, LM-PCR of DNA from cells in normal osmotic medium (nonstress/in vivo); 4, LM-PCR of in vitro methylated DNA (G). Horizontal nucleotide sequence shows the primer 2 location; dotted line indicates the ExoIII stop location and short solid lines indicate guanosine band pattern. Arrows A and B indicate the positions of methylation- protected guanosines bands. Bold underline G indicates the same protected guanosines in the promoter sequence.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: In Vivo, DNA Footprinting, Activity Assay, Cell Culture, In Vitro, Methylation, Sequencing

Journal: The Journal of biological chemistry

Article Title: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter.

doi: 10.1074/jbc.272.51.32500

Figure Lengend Snippet: FIG. 4. EMSA of the 32-bp exonuclease III-protected region with whole cell extract from rat liver cells. The ExoIII-protected 32-bp region was used as a probe to confirm the factor binding and to determine the effect of protein binding by mutation. Lane 1, probe A (no extract added); lane 2, probe A/normal osmolarity (A/n); lane 3, probe A/hyper-osmolarity for 30 min (A/s(309)); lane 4, probe A/hyper-osmolarity for 3 h (A/s(3 h)); lane 5, probe A/normal osmolarity/100 3 cold probe A (A/n comp:A); lane 6, probe A/hyper-osmolarity for 30 min/100 3 cold probe A (A/s(309) comp:A); lane 7, probe A/hyper-osmolarity for 3 h/100 3 cold probe A (A/s(3 h) comp:A); lane 8, probe A8/normal osmolarity (A8/n); lane 9, probe A8 hyper-osmolarity for 30 min (A8/s(309); lane 10, probe A8 hyper-osmolarity for 3 h (A8/s(3 h)); lane 11, probe A/normal osmolarity/100 3 cold probe A8 (A/n comp:A8); lane 12, probe A/hyper-osmolarity for 30 min/100 3 cold probe A8 (A/s(309) Comp:A8); lane 13, probe A/hyper- osmolarity for 3 h/100 3 cold probe A8 (A/s(3 h) Comp:A8). Arrows A-F indicate the complexes. Probe A contains the ExoIII-protected 32-bp region, Probe B is the same as Probe A except for 8 nucleotide replacements. Protected guanosines by dimethyl sulfate methylation (Fig. 3) in Probe A sequence are indicated by an asterisk and the mutations made for probe A8 are indicated by underline. Samples in lanes 1–7 and 11–13 were incubated with probe A.

Article Snippet: Cell Culture, Transfection, and Osmotic Shock—A normal rat liver cell line (Clone 9, ATCC, Rockville, MD) which expresses AR and responds to osmotic stress (data not shown) was cultured in 35-mm * The costs of publication of this article were defrayed in part by the payment of page charges.

Techniques: Binding Assay, Protein Binding, Mutagenesis, Methylation, Sequencing, Incubation

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia

doi: 10.1164/rccm.201906-1232OC

Figure Lengend Snippet: Polyethylenimine-(5) myristic acid/polyethylene glycol oleic acid/cholesterol (PEI600-MA5/PEG-OA/Cho) nanoparticles efficiently target endothelial cells in the neonatal lung. (A) Fluorescence-activated cell sorter (FACS) gating strategy to identify hematopoietic cells (Hema; CD45+CD31−), endothelial cells (Endo; CD31+CD45−CD326−), epithelial cells (Epi; CD326+CD45−CD31−), pericytes (Peric; NG2+PDGFRb+CD45−CD31−CD326−), and myofibroblasts (Myofibro; PDGFRa+CD45−CD31−CD326−). DyLight 650–labeled nanoparticles were delivered at Postnatal Day 2 (P2). FACS analysis of enzymatically digested lung tissue was performed at P5. (B and C) Dot plots show the presence of nanoparticles in different populations of pulmonary cells. Noninjected mice were used as control animals to identify cells containing nanoparticles. (D) Percentage of nanoparticle-targeted cells is shown among pericytes; myofibroblasts; and epithelial, endothelial, and hematopoietic cells (n = 4 mice per group). Error bars are mean ± SE. NG2 = melanoma-associated chondroitin sulfate proteoglycan 4; PDGFRα = platelet-derived growth factor receptor-α; PDGFRβ = platelet-derived growth factor receptor-β.

Article Snippet: Cells not expressing any of these markers (CD45 – CD31 – CD326 – ) were then evaluated for pericytes (PDGFRb + NG2 + CD45 – CD31 – CD326 – ) using PDGFRb (platelet-derived growth factor receptor-β) (CD140b) Ab (clone APB5; BioLegend) and NG2 (melanoma-associated chondroitin sulfate proteoglycan 4) Ab (clone 9.2.27; BD Biosciences).

Techniques: Fluorescence, Labeling, Derivative Assay