Journal: International Journal of Molecular Sciences

Article Title: Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection

doi: 10.3390/ijms22020743

Figure Lengend Snippet: Evaluation of DC-SIGN mediated trans infection among H5N1-PVs carrying N-glycosylation mutations. ( A ) The scheme of modified conventional capture assay is demonstrated. ( B ) Raji and Raji-DC-SIGN were used as captured cells. They were incubated with H5N1-PVs at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three to five times) and the target MDCK cells were subjected to luminescence analysis. In addition, for detecting the virions budding from cis infection, the transwell system was used to monitor those virions released from captured cells further causing MDCK (target cells) infection. The lower channel of infected MDCK cells in the transwell were also subjected to luminescence analysis. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. ( C ) The Raji and Raji-DC-SIGN cells (captured cells) were incubated with H5N1-PVs carrying different N-glycosylation mutations on HA at 4 °C for 2 h and then co-cultured with MDCK (target cells) at 37 °C for 24–48 h. After co-culturing, the capture cells were removed via intensive PBS washing (three-five times) and the target MDCK cells were subjected to luminescence analysis. Similarly, the detection of the virions released from cis infection of the captured cells was monitored using transwell system mentioned above. Alternatively, some groups were co-treated with IgG control and anti-DC-SIGN monoclonal antibodies. The relative infectivity was measured by using the luminescence values of co-cultured MDCK, normalized with values of MDCK from transwell system. The significant difference was measured by each N-glycosylation mutant compared to WT group. Representative results are shown. Quantitative data represent the means ± SD of results from at least three independent experiments (WT, wild-type) (* p < 0.05; ** p < 0.01).

Article Snippet: For a DC-SIGN-enhanced infectivity assay, a 5 × 10 5 susceptible cells mentioned above were seeded into 48-well plates prior to incubation with H5N1 pseudotyped or H5N1-RG virus particles at 37 °C for 2 h. Alternatively, some of these cells were pretreated with anti-DC-SIGN monoclonal antibodies (10 μg/mL-1; R&D System, catalog no. MAB161).

Techniques: Infection, Glycoproteomics, Modification, Incubation, Cell Culture, Control, Bioprocessing, Mutagenesis

Journal: Journal of Cardiothoracic Surgery

Article Title: POLR1D silencing suppresses lung cancer cells proliferation and migration via inhibition of PI3K-Akt pathway

doi: 10.1186/s13019-024-02791-y

Figure Lengend Snippet: Expression of POLR1D in lung cancer cells. a The mRNA expression of POLR1D in human embryonic lung fibroblasts (HFL1) and five-lung cancer cell lines (H2170, SK-MES-1, H226, PC-9, H1975) by RT‑qPCR. b The representative bands of POLR1D protein in six-lung cancer cell lines. c Quantification of POLR1D protein expression determined by Western blot. Data represent the average of three independent experiments (mean ± SD). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. HFL1 group

Article Snippet: Human embryonic lung fibroblasts (HFL1) and lung cancer cell lines (H2170, H226, SK-MES-1, PC-9, and H1975) were obtained from the American Type Tissue Culture Collection (ATCC; Rockville, MD, USA).

Techniques: Expressing, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: MCM4 acts as a biomarker for LUAD prognosis

doi: 10.1111/jcmm.17819

Figure Lengend Snippet: Elevated expression of MCM4 was observed in LUAD tissues and cells, which caused low overall survival rate. (A) MCM4 expression in TCGA datasets. MCM4 level in LUAD tumour samples was higher than that of adjacent normal tissues. (B) The overall survival rate of patients with high MCM4 expression was reduced compared with that with low MCM4 expression. (C) Cox univariate regression analysis of age and histological grade in TCGA cohort. (D) Western blotting assay showed MCM4 level was increased in tumour tissues in relative to those in normal tissues in LSL‐Kras G12D/+ mice model. (E) MCM4 was overexpressed in LUAD cells (H441, H460 and H552) compared with human embryonic lung fibroblast cells (MRC5 and WI38). * p < 0.05. N, adjacent normal tissues; T, tumour tissues. (F) Quantitative results of MCM4 expression.

Article Snippet: Four human LUAD cell lines (H441, H460, H522, A549) and two human embryonic lung fibroblast cell lines (WI38 and MRC5) were obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot