cell lines l929 cells atcc ccl 1 mdck cells watanabe  (ATCC)

Journal: ACS Omega

Article Title: Comparative Study of Chitosan-Pyrophosphate and Magnesium Hydroxide-Alginate Hybrid Nanoparticles: Physicochemical Properties and Cytocompatibility toward Vascular Calcification Applications

doi: 10.1021/acsomega.5c12883

Figure Lengend Snippet: (a) Cytotoxicity analysis of L929 cells after 24 h of exposure to NPs-Mg­(OH) 2 -Alg at different concentrations. * p < 0.05 vs control; (b) cytotoxicity analysis of L929 cells after 24 h of exposure to NPs-CS-PPi at different concentrations. * p < 0.05 vs control.

Article Snippet: The murine fibroblast cell line L929 (ATCC NCTC clone 929 [L cell, CCL-1]) was used for cytotoxicity assays.

Techniques: Control

Journal: PLOS Biology

Article Title: The non-vesicular cholesterol transporter GRAMD1C is a pan-coronavirus antiviral target

doi: 10.1371/journal.pbio.3003736

Figure Lengend Snippet: (A) Genomic sequence analysis of GRAMD1C in WT cells and GRAMD1C-KO PK-15 cells. (B) western blot analysis validated the protein expression level of endogenous GRAMD1C in WT cells and KO cells. (C) GRAMD1C-KO and WT cells were infected with TGEV (MOI = 0.1) and harvested at 24 hpi for western blot analysis. GAPDH used as an internal control gene. (D) The copy number of the TGEV N gene was determined in GRAMD1C-KO and WT cells at 24 hpi, following infection with TGEV (MOI = 0.1), using absolute quantitative real-time PCR. (E) Multiple-step viral growth assay. The viral titers in GRAMD1C-KO and WT cells infected with TGEV (MOI = 1) were monitored at different time points (12, 24, 36, and 48 h). (F) GRAMD1C-KO and WT cells were infected with TGEV at different MOIs (0.01, 0.1, and 1). Immunofluorescence assays were used to detect the TGEV N protein expression at 24 hpi. Scale bar, 400 µm. (G) Overexpression of GRAMD1C in PK-15 control cells at 24 hpi post-TGEV infection (MOI = 0.01). RT-qPCR and viral titer assays for determination of the TGEV replication. WT, transfection of the pCAGGS-HA empty vector in PK-15 cells; WT-overexpression, transfection of the pCAGGS-GRAMD1C-HA vector in PK-15 cells. (H) WT and GRAMD1C-KO Vero-E6 cells were infected with PEDV at 0.1 MOI. Immunofluorescence assays were used to detect the PEDV N protein expression at 24 hpi. Scale bar, 400 µm. The virus titers were tested at 24 hpi. (I) WT and GRAMD1C-KO Caco-2 cells were infected with HCoV-229E at 0.1 MOI. Immunofluorescence assays were used to detect the HCoV-229E N protein expression at 36 hpi. Scale bar, 200 µm. The virus titers were tested at 36 hpi. (J) WT and GRAMD1C-KO Vero-E6 cells were infected with SARS-CoV-2 at 0.01 MOI. Immunofluorescence assays were used to detect the SARS-CoV-2 N protein expression at 24 hpi. Scale bar, 200 µm. The virus titers were tested at 24 hpi. Western blot analysis for SARS-CoV-2 N protein at 24 hpi (MOI = 0.1). GAPDH used as an internal control gene. (K) WT and GRAMD1C-KO L929 cells were infected with MHV (MOI = 0.01). MHV titers were tested at 12 hpi. WT cells exhibited pronounced cytopathic effects compared to KO cells. (L) WT and GRAMD1C-KO PK-15 cells were infected with PDCoV at 5 MOI in the presence of 7.5 μg/ml trypsin. Immunofluorescence assays were used to detect the PDCoV N protein expression at 24 hpi. Scale bar, 400 µm. PDCoV titers were tested at 24 hpi. The means and SDs of the results from three independent experiments are shown. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. P -values were determined by two-tailed Student’s t -tests. The data underlying this Figure can be found in and .

Article Snippet: Vero-E6 (C1008) and L929 (CCL-1) cell lines were purchased from ATCC (USA).

Techniques: Sequencing, Western Blot, Expressing, Infection, Control, Real-time Polymerase Chain Reaction, Growth Assay, Immunofluorescence, Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Virus, Two Tailed Test

Journal: PLOS Biology

Article Title: The non-vesicular cholesterol transporter GRAMD1C is a pan-coronavirus antiviral target

doi: 10.1371/journal.pbio.3003736

Figure Lengend Snippet: (A) Schematic of 20α-HC-mediated blockade of cholesterol transport by the GRAMD1C ASTER domain from PM to ER. (B) PK-15 cells were incubated with different concentrations of 20α-HC in advance for 8 hours, and then cells were infected with TGEV (MOI = 0.1). Subsequently, the viral titer assay was performed. (C) The TGEV N protein was detected by immunofluorescence assays with different concentrations of 20α-HC. Scale bar, 400 µm. (D) PK-15 cells were infected with TGEV (MOI = 0.1) and 20α-HC was added after viral entry for 8 hours. The TGEV titers were tested at 24 hpi. The TGEV N copy number was assessed by RT-qPCR assay at 24 hpi. (E) Confocal microscopy to evaluate TGEV replication by detecting dsRNA formation in cells treated with 20α-HC after virus entry. The cells were infected with TGEV (MOI = 1) for 12 hours. Scale bars, 10 µm. (F) Co-localization of TGEV-nsp3-HA (green) and TGEV-nsp4-His (red) was analyzed by confocal microscopy in HEK-293T cells with or without inhibitor treatment for 8 hours, after transfection. Scale bar, 10 μm. (G and H) TEM analysis demonstrated the presence of DMVs in WT and inhibitor-treated cells following the overexpression of plasmids encoding nsp3 and nsp4. DMVs were manually counted within complete single-cell profiles ( n = 5 cell profiles per group). Scale bar, 2 µm. (G) HEK293T cells overexpressed with nsp3 and nsp4. Atypical crescent-shaped structures in inhibitor-treated cells are denoted by red arrows. (H) PK-15 cells overexpressed with nsp3 and nsp4. (I) Caco-2 cells were infected with HCoV-229E (MOI = 0.1), and then cells were incubated with 10µM 20α-HC for 8 hours. The HCoV-229E N protein and virus titers were detected at 36 hpi. (J) Vero-E6 cells were infected with SARS-CoV-2 (MOI = 0.01), and then cells were incubated with 30µM 20α-HC for 8 hours. The SARS-CoV-2 N protein and virus titers were detected at 24 hpi. (K) Vero-E6 cells were infected with PEDV (MOI = 0.1), and then cells were incubated with 30µM 20α-HC for 8 hours. The PEDV titers were tested at 24 hpi. PK-15 cells were infected with PDCoV (MOI = 5) in the presence of 7.5 μg/ml trypsin, and then cells were incubated with 30µM 20α-HC for 8 hours. The PDCoV titers were tested at 24 hpi. L929 cells were infected with MHV (MOI = 0.01), and then cells were incubated with 30µM 20α-HC for 8 hours, The MHV titers were tested at 18 hpi. (L) The proposed model of the roles of GRAMD1C in DMV biogenesis. During the coronavirus replication stage, the viral nsp3 and nsp4 induce zippering and bending of the ER. In this process, nsp4 recruits GRAMD1C to DMV formation sites by binding to its TM domain, utilizing cholesterol delivered to the ER by GRAMD1C for DMV biogenesis. In GRAMD1C-KO cells or inhibitor-treated cells, insufficient cholesterol supply at DMV formation sites results in the generation of only crescent-shaped vesicular structures, thereby inhibiting DMV formation. The means and SDs of the results from three independent experiments are shown. ns, not significant; ** P < 0.01; *** P < 0.001; **** P < 0.0001. P -values were determined by two-tailed unpaired Student’s t -tests. The da t a underlying this Figure can be found in .

Article Snippet: Vero-E6 (C1008) and L929 (CCL-1) cell lines were purchased from ATCC (USA).

Techniques: Incubation, Infection, Titer Assay, Immunofluorescence, Quantitative RT-PCR, Confocal Microscopy, Virus, Transfection, Over Expression, Single Cell, Binding Assay, Two Tailed Test

Journal: PLOS Biology

Article Title: The non-vesicular cholesterol transporter GRAMD1C is a pan-coronavirus antiviral target

doi: 10.1371/journal.pbio.3003736

Figure Lengend Snippet: (A) Line graphs showing the body weight of GRAMD1C +/− and WT mice during the 3 days post-inoculation with MHV A59 ( n = 6 mice per genotype). (B) The total weight change of GRAMD1C +/− and WT mice during the 3 days post-inoculation of MHV A59 ( n = 6 mice per genotype). (C) Survival curves for GRAMD1C +/− and WT mice infected with MHV A59 ( n = 6 mice per genotype). (D and E) Histopathological analysis of the degree of liver damage in WT and GRAMD1C +/− mice infection with MHV A59 at 3 dpi. (D) Gross postmortem examination of liver tissue; (E) histological examination of Hematoxylin–eosin (H&E) stained liver tissue. The lesion area is indicated by the arrow. Scale bar, 100 µm. (F) IHC staining for dsRNA in the liver tissue of WT and GRAMD1C +/ mice at 3 dpi following infection with MHV A59 (positive signals indicated by arrowheads). Scale bar, 100 µm. (G) The virus titers of half livers of GRAMD1C +/− and WT mice were measured TCID 50 assay with L929 cells ( n = 3 mice per genotype). (H) Schematic diagram of the mouse inhibitor experiment. WT Mice were administered 20 mg/kg 20α-HC via intraperitoneal injection 24 hours and 12 hours before, as well as after viral challenge. (I) Line graphs showing the body weight of 20α-HC-treated and WT mice during the 3 dpi with MHV A59 ( n = 6 mice per genotype). (J) The total weight change of 20α-HC-treated and WT mice during the 3 days post-inoculation of MHV A59 ( n = 6 mice per genotype). (K) Survival curves for WT and 20α-HC-treated mice infected with MHV A59 ( n = 6 mice per genotype). (L and M) Histopathological analysis of the degree of liver damage in WT and 20α-HC-treated mice infection with MHV A59 at 3 dpi. (L) Gross postmortem examination of liver tissue; (M) histological examination of Hematoxylin–eosin (H&E) stained liver tissue. Scale bar, 100 µm. (N) IHC staining for dsRNA in the liver tissue of WT and 20α-HC-treated mice at 3 dpi following infection with MHV A59 (positive signals indicated by arrowheads). Scale bar, 100 µm. (O) The virus titers of half livers of 20α-HC treated and WT mice were measured TCID 50 assay with L929 cells ( n = 3 mice per genotype). The means and SDs of the results from three independent experiments are shown. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. P -values were determined by two-tailed unpaired Student’s t -tests. The da t a underlying this Figure can be found in .

Article Snippet: Vero-E6 (C1008) and L929 (CCL-1) cell lines were purchased from ATCC (USA).

Techniques: Infection, Staining, Immunohistochemistry, Virus, Injection, Two Tailed Test