Journal: Glycobiology

Article Title: Identification of C -mannosylation in a receptor tyrosine kinase AXL

doi: 10.1093/glycob/cwae096

Figure Lengend Snippet: DPY19 family proteins are not C -mannosyltransferases of AXL. (A) Establishment of AXL-ECD and DPY19 family-co-expressing S2 cells. S2 cells were transfected with pMT-PURO-AXL-ECD and pIZ-DPY19L1, L2, L3, or L4. mRNA expression levels of DPY19 family were measured by RT-PCR. Mock S2 cells were co-transfected with pMT-PURO-AXL-ECD and pIZ/V5-his empty vector. (B) C -mannosylation of AXL is not catalyzed by DPY19 family proteins. Recombinant AXL-ECD was purified from conditioned media of DPY19 family-expressing S2 cells. These samples were analyzed by LC–MS/MS. Only unmodified 309 VACTSSQGPSSWTHWLPVETPE 330 peptides were detected in samples from mock and DPY19L1-, L2-, L3-, and L4-expressing cells as m / z = 1235.6. (C and D) mRNA expression level of DPY19 family in MDA-MB-231 cells. cDNA of HT1080 and NEC8 cells were used as a positive control of DPY19L1, L3, and L4 (HT1080, C), and DPY19L2 (NEC8, D).

Article Snippet: Human NSCLC PC9 (American Type Culture Collection, Manassas, VA) and H3122 cells (gifted by Dr. JA Engelman, Massachusetts General Hospital Cancer Center, Boston, MS), and human embryonal carcinoma NEC8 cells (RIKEN BioResource Center) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal bovine serum, 100 mg/L kanamycin, 100 units/mL penicillin G, 300 mg/L L-glutamine, and 2.25 g/L NaHCO 3 , at 37 °C with 5% CO 2 .

Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Purification, Liquid Chromatography with Mass Spectroscopy, Positive Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human sapovirus propagation in human cell lines supplemented with bile acids

doi: 10.1073/pnas.2007310117

Figure Lengend Snippet: Human cell lines NEC8 and HuTu80 but not HCT-8 supported the replication of HuSaV GI.1 and GII.3 in the presence of bile acids in the cell culture medium. HuSaV GI.1 (AK20) or GII.3 (AK11) positive stool suspension was inoculated onto the cell monolayers in the presence of CA, GlyCA, DCA, GCDCA, or Bile, or in the absence of bile acids (Mock). After removing the inoculum and washing the cell monolayers at 1 dpi, we collected the culture supernatants at 7 dpi. The optimal concentration of bile acid for each cell line, as indicated in Materials and Methods, was present throughout the culture period. The HuSaV RNA was measured by RT-PCR followed by agarose gel electrophoresis. The GI.1 inoculum and water was included as positive and negative controls, respectively, for HCT-8 cells. The results were confirmed in an independent experiment, and pictures of a representative experiment are shown.

Article Snippet: For HuSaV culture trials, we used three human cell lines: 1) human testicular embryonal carcinoma-derived cell line, NEC8 cells (Japanese Collection of Research Bioresources [JCRB] 0250); 2) human duodenum carcinoma derived cell line, HuTu80 cells (American Tissue Culture Collection [ATCC] #HTB-40); and 3) human ileocecal adenocarcinoma derived cell line, HCT-8 (HRT-18) cells (ATCC #CCL-244).

Techniques: Cell Culture, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Human sapovirus propagation in human cell lines supplemented with bile acids

doi: 10.1073/pnas.2007310117

Figure Lengend Snippet: Growth kinetics of HuSaV GI.1 and GII.3 in NEC8 and/or HuTu80 cells. HuSaV GI.1 (AK20) or GII.3 (AK11) positive stool suspension was inoculated onto NEC8 or HuTu80 cell monolayers. After removing the inoculum and washing the cell monolayers at 1 dpi, we collected supernatants at 1, 3, 5, and 7 dpi or 1, 3, 5, 7, 10, and 14 dpi. GlyCA (500 µM for NEC8 and 1,000 µM for HuTu80) was present throughout the culture period. The SaV RNA was measured by RT-qPCR. Dotted line indicates the lowest quantification limit. Data represent the values of two independent experiments using cells at different passage numbers for each time point. Error bars indicate SDs.

Article Snippet: For HuSaV culture trials, we used three human cell lines: 1) human testicular embryonal carcinoma-derived cell line, NEC8 cells (Japanese Collection of Research Bioresources [JCRB] 0250); 2) human duodenum carcinoma derived cell line, HuTu80 cells (American Tissue Culture Collection [ATCC] #HTB-40); and 3) human ileocecal adenocarcinoma derived cell line, HCT-8 (HRT-18) cells (ATCC #CCL-244).

Techniques: Quantitative RT-PCR