Journal: Gates Open Research

Article Title: Analytical performance and clinical usability of a low-cost vaginal swab for sensitive detection of high-risk human papillomavirus

doi: 10.12688/gatesopenres.16399.1

Figure Lengend Snippet: Limit of Detection of different HPV genotypes on the Steripack XpressCollect flocked nylon swab on the Atila ScreenFire HPV RS assay.

Article Snippet: Human cervical cancer cell lines including SiHa (ATCC HTB-35), HeLa (ATCC CCL-2), CaSki (ATCC CRL-1550), and C-33A (ATCC HTB-31) cells from American Type Culture Collection (ATCC, Manassas, VA, USA) were grown at 37°C with 5% CO 2 in culture media: Dulbecco’s Modified Eagle Medium high glucose with sodium pyruvate containing 10% Fetal Bovine Serum (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 50 μg/mL gentamycin (Sigma-Aldrich, St. Louis, MO, USA) under standard aseptic conditions while screening for mycoplasma contamination using the Universal Mycoplasma Detection kit (ATCC, Manassas, VA, USA) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation

Journal: Science Advances

Article Title: Phosphorylation by CaMKIV regulates the transcriptional repressor activity and half-life of REST/NRSF

doi: 10.1126/sciadv.aec0781

Figure Lengend Snippet: ( A ) Schematics of the four possible degradation pathways that CaMKIV-phosphorylated REST can undertake and the three inhibitors that were used to dissect the fate of REST. Cat, cathepsin. ( B ) HEK293T cells were transfected with WT, CA, or KD CaMKIV and treated with cycloheximide (CHX) to in4hibit further translation. Then, cells were treated for 16 hours with either vehicle [dimethyl sulfoxide (DMSO)] or MG-132 (5 μM), an inhibitor of the ubiquitin-proteasome system (UPS). Left: Representative immunoblots monitoring the expression of WT, CA, and KD CaMKs, REST, and calnexin, respectively, in HEK293T cells, 48 hours after transfection with the kinase constructs. Right: Mean (±SEM) REST protein levels, shown as ratios with calnexin immunoreactivity under the various experimental conditions. No effects of inhibition of the UPS were observed. * P = 0.017, CA versus WT in DMSO; ** P = 0.0048, CA versus WT in MG-132. Two-way ANOVA/Tukey’s tests ( n = 4 independent preparations). ( C ) HEK293T cells were transfected with either WT or CA CaMKIV and treated with CHX to inhibit further translation. Successively, cells were treated for 16 hours with DMSO (Veh), bafilomycin A1 (Baf; 100 nM; an inhibitor of lysosomal acidification and autophagy), leupeptin (Leu; 40 μM; an inhibitor of the lysosomal and cytosolic proteases), CA074 (a specific inhibitor of Cathepsin B), or E64 (an inhibitor of cathepsins). Left: Representative immunoblots monitoring the expression of REST, calnexin, and the autophagy markers p62 and LC3B-I and -II. Right: Mean (±SEM) REST/calnexin ratios normalized with respect to the control REST/calnexin ratio in WT CaMKIV–expressing cells. Leu, * P = 0.0351; CA074, * P = 0.0361; E64, * P = 0.0481. Mixed-effects ANOVA/Dunnett’s tests ( n = 4 independent preparations).

Article Snippet: W7, KN92, KN93, CA074, E64, bicuculline, CGP58845 , d -aminophosphovalerate, and tetrodotoxin were from Tocris (Bristol, UK).

Techniques: Transfection, Ubiquitin Proteomics, Western Blot, Expressing, Construct, Inhibition, Control