Journal: mBio

Article Title: G521 is the gatekeeper and a key transmembrane domain contact residue of Candida albicans Cdr1

doi: 10.1128/mbio.03746-25

Figure Lengend Snippet: ( A ) SDS-PAGE of 20 μg crude PM proteins from AD cells overexpressing 11 Cdr1-G521 variants, including wild-type Cdr1 and the negative control strain AD/pABC3. Arrows indicate the 170 kDa Cdr1 and the 100 kDa Pma1 bands. M = molecular weight markers. The % Cdr1 expression levels relative to wild-type Cdr1 are listed underneath the images. ( B ) Heatmap of log 2 -transformed fold-increased (blue) or -decreased (red) MIC values of 11 antifungals for AD strains overexpressing wild-type Cdr1 (the reference AD/CDR1B-G521 strain) or 10 different Cdr1-G521 variants. Strains and antifungals are ordered from top to bottom and from left to right according to their molecular weights, which are given in brackets. A color scale depicting the range of fold-increased (0 to 2 = wild-type level to fourfold increased) and fold-decreased (0 to −9 = wild-type level to 512-fold decreased) MIC values is shown to the right of the heatmap. CER: cerulenin, CHX: cycloheximide, FLC: fluconazole, VRC: voriconazole, MCZ: miconazole, R6G: rhodamine 6G, KTC: ketoconazole, MON: monensin, POS: posaconazole, ITC: itraconazole, NIG: nigericin. ( C ) Glucose-dependent R6G efflux by whole cells expressing the 10 Cdr1-G521 variants or wild-type Cdr1. Extracellular R6G fluorescence was measured at 30 s intervals post glucose addition (t = 0). Data are the means of two biological replicates. The R6G efflux values were normalized by (i) subtracting the background signal of the negative control strain AD/pABC3, (ii) adjusting for the slightly different Cdr1 expression levels, and (iii) setting the extracellular R6G levels obtained by Cdr1-G521-expressing cells after 30 min incubation in the presence of glucose to 100%. The molecular weights (Da) of the residues at position 521 are provided in brackets next to individual residues. ( D ) Graph of the maximum R6G efflux rates of whole cells of the positive control strain AD/CDR1B (blue circles) and the R521 (green triangles) and H521 (red circles) variants plotted against the R6G concentrations used to load the cells before initiating R6G efflux from cells with the addition of glucose. The data represent the means ± SD of three biological replicates. Kinetic parameters V max and K half for each strain are tabulated adjacent to the figure.

Article Snippet: The following chemicals were purchased: MON, OLI, VAN, BEA, and 2-Deoxy-D-glucose (2-DOG) from Sigma-Aldrich; MCZ, NIG, CER, and R6G from FUJIFILM Wako Pure Chemical Corporation; POS and ITC from Tokyo Chemical Industry; VRC and CHX from NACALAI TESQUE; FLC and KTC from LKT Laboratories.

Techniques: SDS Page, Negative Control, Molecular Weight, Expressing, Transformation Assay, Fluorescence, Incubation, Positive Control

Journal: European Journal of Dentistry

Article Title: Effects of Different Disinfection Methods on Microleakage of Giomer Restorations

doi: 10.1055/s-0039-1698370

Figure Lengend Snippet: Disinfection methods used in the study

Article Snippet: Group 1 , CHX 2% chlorhexidine gluconate treatment with rinsing and drying , Cavity Cleanser, Bisco, Schaumburg, Illinois, United States.

Techniques:

Journal: Cell Death and Differentiation

Article Title: E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53

doi: 10.1038/cdd.2011.57

Figure Lengend Snippet: Hades reduces the cytoplasmic p53 level. (a) Immunoblot analysis showed that ectopically expressed Hades reduces the level of endogenous p53. U2OS cells were transfected with increasing amounts of GFP-Hades plasmid. At 24 h after transfection, the level of p53 protein was examined by immunoblot analysis. (b) Hades decreases the level of ectopically expressed p53, depending upon its ubiquitin ligase activity. H1299 cells were cotransfected with 1 μg of each indicated plasmid for 24 h and treated with 10 μM MG132 for 4 h. (c) Generation of stable Hades-knockdown MCF7 cells using the shRNA, pSuper-Hades. After selection for 14 days in media containing G418 (500 μg/ml), hades mRNA levels were measured by RT-PCR and the levels of endogenous Hades and p53 proteins were measured by immunoblotting. (d) Ablation of Hades increases p53 stability. Stable Hades-knockdown MCF7 cells were incubated with cycloheximide (CHX, 150 μg/ml) for the indicated times. Endogenous levels of p53 were measured by immunoblotting, while residual cellular levels of p53 were quantitated by densitometry. The half-life of p53 was measured based on the decay of normalized (to Actin control) p53 levels to 50% of their original level. Values are an average of two independent experiments. (e) Hades-mediated degradation of p53 is recovered in the presence of leptomycin B (LMB). H1299 cells were cotransfected with 1 μg of each indicated plasmid. After 24 h, cells were treated with LMB (20 nM), for 4 h (lanes 3–6), and the level of p53 and GFP-Hades was measured by immunoblotting using anti-p53 and anti-GFP antibodies. An anti-Actin antibody was used as a loading control. (f) p53 stability in H1299 cells cotransfected with 0.5 μg wild-type or mutant (C135Y) p53 plasmid and the indicated amounts of GFP-Hades plasmid. At 24 h after transfection, ectopically expressed p53 and GFP-Hades were measured by immunoblotting. In addition, stability was determined as in (d). Values are the average of two independent experiments

Article Snippet: E1, E2 family, and His-ubiquitin were from Boston Biochem (Cambridge, MA, USA); CPT, Actinomycin D, crystal violet, propidium iodine, and DAPI from Sigma; CHX from Biopure (Burlington, Ontario, Canada); MG132 from Calbiochem (La Jolla, CA, USA); the JC-1 staining kit from Cayman Chemical (Ann Arbor, MI, USA); Annexin V from BD Pharmingen; and LMB from Alexis Biochemical (San Diego, CA, USA).

Techniques: Western Blot, Transfection, Plasmid Preparation, Activity Assay, shRNA, Selection, Reverse Transcription Polymerase Chain Reaction, Incubation, Mutagenesis