Journal: Pharmaceutical Research

Article Title: Cellular Uptake of Cationic Polymer-DNA Complexes Via Caveolae Plays a Pivotal Role in Gene Transfection in COS-7 Cells

doi: 10.1007/s11095-007-9287-3

Figure Lengend Snippet: Uptake ( a ) and transfection ( b ) of polyplexes in time. COS-7 cells were incubated with pDMAEMA- or PEI-based polyplexes for 60 minutes at 4°C. Subsequently, the cells were incubated at 37°C and after different incubation times the internalized fluorescence ( a ) or the luciferase expression was determined ( b ). The open bars represent pDMAEMA- and the filled bars PEI-incubated cells. Results are means ± SD from two different experiments.

Article Snippet: Of the cell suspension 20 μl was diluted in 100 μl luciferase reaction buffer (Promega) and the luminescence measured after 10 s using a luminometer (Berthold).

Techniques: Transfection, Incubation, Fluorescence, Luciferase, Expressing

Journal: mSphere

Article Title: Construction of Candida albicans Strains with ATP-Analog-Sensitive Protein Kinase A and Hog1

doi: 10.1128/msphere.00095-23

Figure Lengend Snippet: Inhibition of AS Tpk1 and Tpk2 by 1NM-PP1 blocks C. albicans growth and hyphal morphogenesis. (A) A flowchart outlining the strain construction strategy used to make the TPK1 and TPK2 AS mutants. A black arrow indicates deletion or complementation at both alleles using a transient CRISPR strategy . Two orange arrows indicate the sequential complementation of both alleles using homologous recombination. The marker code utilizes gray to indicate auxotrophy for the indicated amino acid or sensitivity to the indicated drug. Red indicates prototrophy for the amino acid or resistance to the drug indicated. (B) Treatment with 1NM-PP1 inhibits the growth of AS TPK1 or TPK2 strains. C. albicans strains were grown overnight to saturation in YPD and then diluted to an optical density at 600 nm (OD 600 ) of 0.002 into fresh YPD containing either 2.5 μM 1NM-PP1 or dimethyl sulfoxide (DMSO) vehicle control. Strains were grown at 30°C for 24 h and growth was assessed by OD 600 (see color bar). (C) Treatment with 2.5 μM 1NM-PP1 inhibits filamentation of the AS TPK2 strain. Strains were grown at 37°C in either YPD with 10% serum; Spider medium; or yeast nitrogen base supplemented with 11 mM glucose, 2% casamino acid, and 5 mM GlcNAc (YNBNAG) for 4 h. (D) Treatment with 5 μM 1NM-PP1 inhibits filamentation of the AS TPK1 and TPK2 strains in RPMI with 10% serum and 5% CO 2 at 37°C. Strains were grown for 4 h. (E) TPK1 expression and TPK2 expression are not significantly different in wild-type or AS C. albicans strains. Transcript levels were normalized to ACT1 . Data are presented as mean ± SD of technical triplicates. (F) A strain of C. albicans with both AS TPK1 and TPK2 alleles exhibited analog-specific inhibition of growth. C. albicans strains were grown as described in B. (G) Treatment with 1NM-PP1 inhibits filamentation in a strain of C. albicans with both AS TPK1 and TPK2 alleles. Strains were grown in YNBNAG at 37°C for 4 h. (H) Silver staining of the purified Tpk2/Bcy1 holoenzyme. Strains were grown to log phase in YPD, and then the PKA holoenzyme was purified using a flag-affinity purification as described previously . HF represents His6-Flag3. The purified product was run on a 10% SDS-PAGE gel and silver stained. *, impurities. (I) PKA kinase assays were performed using the Peptag kit (Promega). The nonphosphorylated peptide contains one positive charge (+1) and migrates toward the cathode. The phosphorylated peptide contains one negative charge (−1) and migrates toward the anode. Products were resolved on a 0.8% agarose gel in 50 mM Tris-HCl (pH 8.0). The gel was imaged with an Alexa 568 channel. In all panels, the acronym AS stands for analog sensitive. All experiments were performed in biological duplicate.

Article Snippet: Using an in vitro PepTag PKA activity assay (Promega), we confirmed that the Tpk2/Bcy1 holoenzymes phosphorylate a fluorescent peptide substrate in the presence of cAMP.

Techniques: Inhibition, CRISPR, Homologous Recombination, Marker, Control, Expressing, Silver Staining, Purification, Affinity Purification, SDS Page, Staining, Agarose Gel Electrophoresis