Project description:Lysosomes are key degradative compartments of the cell. Transport to lysosomes ismediated by tagging soluble enzymes with mannose 6-phosphate (M6P) by GlcNAc-1-phosphotransferase whose deficiency leads to the severe lysosomal storage disorder mucolipidosisII (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thusdepend on functional GlcNAc-1-phosphotransferase. Using genome-scale CRISPR screens, weidentify LYSET as essential for infection by cathepsin-dependent viruses including SARS-CoV-2. We show that LYSET deficiency results in global loss of M6P tagging and mislocalization ofGlcNAc-1-phosphotransferase to lysosomes. Lyset knockout mice exhibit MLII-like phenotypesand human pathogenic LYSET alleles fail to restore lysosomal sorting defects. Thus, we uncoverLYSET as an indispensable component of the M6P trafficking machinery and reveal thebiochemical basis of an inherited disease caused by mutations in LYSET.
Project description:Compared to the Scramble U87 cells, MMP14 ablation induces G2M arrest SABioscience Cell cycle platform, human genes. U87 cells were infected separately with eithr lentivirus carry on Scramble or sh RNA against human MMP14 gene.Equla amount of mRNA was isolated prior RT-PCR analyses
Project description:In the unicellular eukaryote Saccharomyces cerevisiae, Cln3-CDK activity enables Start, the irreversible commitment to the cell division cycle. However, the concentration of Cln3 has been paradoxically considered to remain constant during G1, due to the presumed scaling of its production rate with cell size dynamics. Measuring metabolic and biosynthetic activity during cell cycle progression in single cells, we found that cells exhibit pulses in protein production rate, which do not scale with cell size dynamics, but -following the intrinsic metabolic dynamics- peak around Start. Using a viral-based bicistronic construct and targeted proteomics to measure Cln3 at the single-cell and population level, we show that the differential scaling between protein production and cell size leads to a temporal increase in Cln3 concentration, and passage through Start. This differential scaling causes Start in both daughter and mother cells, across growth conditions. Thus, uncoupling between two fundamental physiological parameters drives cell cycle commitment.
Project description:RT-PCR analysis of inflammatory cytokines and receptors following Il1alpha treatment of pEGP-miR-135b over-expressing cells compard to controls (pEGP-miR-null) using pathway-specific PCR arrays for the evaluation of mouse inflammatory cytokines and receptors (#PAMM-011D). qPCR array gene expression profiling. This experiment examined the transcriptional response of NIH3T3 cells over-expressing pEGP-miR-135b following IL1alpha treatment. Total RNA (n = 3 per group) was used with SABiosciences pathway-specific PCR arrays for inflammatory cytokines and receptors (#PAMM-011D).
Project description:RT-PCR analysis of inflammatory cytokines and receptors in pEGP-miR-135b over-expressing cells compard to controls (pEGP-miR-null) using pathway-specific PCR arrays for the evaluation of mouse inflammatory cytokines and receptors (#PAMM-011D). qPCR array gene expression profiling. This experiment examined the transcriptional response of NIH3T3 cells over-expressing pEGP-miR-135b. Total RNA (n = 3 per group) was used with SABiosciences pathway-specific PCR arrays for inflammatory cytokines and receptors (#PAMM-011D).
Project description:Comparison of PBMC transcriptional profiles in healthy subjects, patients with Crohn's Disease, and patients with Ulcerative Colitis