Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours. Under these conditions EHD1 was seen to co-localize with SMO in the primary cilia. A profile scan for SMO and EHD1 in the primary cilia merged panel is shown. Under these conditions, EHD1 was seen to traffic to the cilia of 60% cells studied. ( B ) WT NIH3T3 cells stably expressing SMO-GFP and transiently expressing EHD1-DsRed were starved in low serum media for 24 hours and stimulated with SAG immediately before starting live imaging of Smoothened and EHD1.As reported in earlier studies, Smoothened was found in preciliary vesicles under non-stimulated conditions but upon SHH pathway activations, EHD1 was seen to associate with Smoothened vesicles and co-traffic with SMO into the primary cilia.

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Stable Transfection, Expressing, Imaging

Journal: Scientific Reports

Article Title: Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

doi: 10.1038/srep20727

Figure Lengend Snippet: ( A ) Immunoblotting was used to assess the presence of SMO in anti-EHD1 immunoprecipitates from WT NIH3T3 cells stably expressing SMO GFP treated with SAG (24 hrs). Input samples show the amount of each protein in the whole extract before the IP. Immunoblots showing the amount of SMO that co-precipitated with EHD1 from cells that stably expressed the protein and were treated with SAG for 24 hrs. A control IP was performed with non-specific rabbit IgG instead of EHD1 antibody and WT NIH3T3 cell lysates stably expressing SMO GFP treated with SAG (24 hrs). Arrows denote the molecular weight fraction of SMO that associates with EHD1. Full-length blots/gels are presented in . ( B ) WT NIH3T3 cells stably expressing SMO-GFP were starved in low serum media for 24 hours and stimulated with SAG in starvation media for another 24 hours and lysed and these lysates were incubated with GST-EHD1 or GST-EH domain deleted EHD1. The membranes were probed with antibodies to Smoothened and GST.GST-fused to the empty plasmid vector was used as a negative control. The data is representative of three separate experimental repeats. Full-length blots/gels are presented in .

Article Snippet: NIH3T3 SMO-GFP cells were generated by transfecting NIH3T3 cells with a SMO-GFP reporter construct (Genecopoeia #MPRM17869-LvPF02) and selecting stable clones after FACS sorting.

Techniques: Western Blot, Stable Transfection, Expressing, Control, Molecular Weight, Incubation, Plasmid Preparation, Negative Control

Journal: Scientific Reports

Article Title: HPV16-Immortalized Cells from Human Transformation Zone and Endocervix are More Dysplastic than Ectocervical Cells in Organotypic Culture

doi: 10.1038/s41598-018-33865-2

Figure Lengend Snippet: HPV16-immortalized cells from TZ and endocervix are more dysplastic. ( a ) HPV16-immortalized cells derived from ectocervix, TZ, or endocervix were maintained for 10 days on rafts with cervical stromal cells. All cell lines were tested at approximately 80 to 95 population doublings. Dotted lines indicate position of basement membrane and the upper extent of epithelial cells. Upper row of figures were stained with H&E and lower row were simultaneously stained for K14, K18 and DAPI. ( b ) Mean dysplastic index (degree of dysplasia) ± standard error of 24 different HPV16-immortalized cell lines (8 from ectocervix, TZ and endocervix) maintained in organotypic culture on rafts formed with 3T3-J2 mouse cells or human stromal cells. Bars above graph show values that are statistically different (3 asterisks = p < 0.001).

Article Snippet: The mouse 3T3-J2 cell line was purchased from Kerafast and cultured in DMEM with 10% FBS plus antibiotics.

Techniques: Derivative Assay, Staining

Journal: Scientific Reports

Article Title: HPV16-Immortalized Cells from Human Transformation Zone and Endocervix are More Dysplastic than Ectocervical Cells in Organotypic Culture

doi: 10.1038/s41598-018-33865-2

Figure Lengend Snippet: HPV16-immortalized cells from TZ and endocervix are more invasive. ( a ) HPV16-immortalized cells derived from ectocervix, TZ, or endocervix (all 85 population doublings) were grown for 10 days on rafts with either 3T3-J2 mouse cells or human stromal cells. Dotted lines indicate position of basement membranes. ( b ) Mean invasion index (degree of invasion into collagen raft) ± standard error of 24 different HPV16-immortalized cell lines (8 from ectocervix, TZ and endocervix) maintained in organotypic culture on rafts formed with 3T3-J2 cells or human stromal cells. Bars show values that are statistically different (3 asterisks = p < 0.001).

Article Snippet: The mouse 3T3-J2 cell line was purchased from Kerafast and cultured in DMEM with 10% FBS plus antibiotics.

Techniques: Derivative Assay

Journal: Microbiology Spectrum

Article Title: BET-Independent Murine Leukemia Virus Integration Is Retargeted In Vivo and Selects Distinct Genomic Elements for Lymphomagenesis

doi: 10.1128/spectrum.01478-22

Figure Lengend Snippet: In vivo replication of WT MLV and W390A MLV in murine tissue. (A) Schematic representation of the workflow to evaluate the in vivo infectivity of WT MLV and W390A MLV. Newborn mice were infected by intraperitoneal injection with 4 × 10 5 RTU of WT MLV or W390A MLV 1 day after birth. Blood was drawn between 80 and 90 days after injection. Extracts of the spleen and thymus were made at 3 and 5 weeks postinfection. (B) MLV loads in spleen cells from mice infected with WT MLV ( n = 5) or W390A MLV ( n = 5) or injected with PBS ( n = 2) at 3 and 5 weeks postinfection. The viral load was measured by RT-qPCR of MLV IN relative to GAPDH levels and represented as relative units (RU). (C) Number of infected NIH 3T3 cells after 1 day of coculture with cells from spleens of mice infected with WT MLV ( n = 4) or W390A MLV ( n = 4) or injected with PBS ( n = 2) at 3 and 5 weeks postinfection. (D) MLV loads in thymus cells from mice infected with WT MLV (3 wpi, n = 2; 5 wpi, n = 3) or W390A MLV (3 wpi, n = 2; 5 wpi, n = 4) or injected with PBS ( n = 2) at 3 and 5 weeks postinfection. The viral load was measured by RT-qPCR of MLV IN relative to GAPDH levels. (E) Number of infected NIH 3T3 cells after 1 day of coculture with cells from spleens of mice infected with WT MLV (3 wpi, n = 2; 5 wpi, n = 3) or W390A MLV (3 wpi, n = 2; 5 wpi, n = 4) or injected with PBS ( n = 2) at 3 and 5 weeks postinfection. No statistically significant difference was found using a Kruskal-Wallis test (B to E). ns, not significant.

Article Snippet: The NIH 3T3 cell line was a kind gift from Johan Van Lint from the Laboratory for Protein Phosphorylation and Proteomics (KU Leuven).

Techniques: In Vivo, Infection, Injection, Quantitative RT-PCR