Journal: Retrovirology

Article Title: The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

doi: 10.1186/1742-4690-10-29

Figure Lengend Snippet: Mutations at the NTD-CTD intersubunit interface impair HIV-1 infectivity and alter capsid stability. ( A ) Production of viral particles as measured by RT activity. ( B ) Immunoblot analysis of viral lysates. Virions were collected from transfected 293T cells, pelleted, and analyzed by SDS-PAGE and immunoblotting with a CA specific polyclonal antibody. ( C ) Single-cycle infectivity of the mutant virions. Virion stocks were titrated on TZM-bl reporter cells and the extent of infection was determined by quantification of luciferase reporter activity in cell lysates. Values were normalized by the corresponding values of reverse transcriptase activity in the inocula. Results shown are the mean values of three independent experiments, with error bars representing one SD. ( D ) Quantification of core-associated CA. Cores were purified from the concentrated virions, and the levels of the core-associated CA were determined as a percentage of the total virion-associated CA. The values shown are the means of three independent experiments; error bars represent one SD.

Article Snippet: For analysis of viral DNA synthesis by qPCR, viruses were produced in 293T cells using the 293T transfection reagent (Mirus), according to the manufacturer’s protocol.

Techniques: Infection, Activity Assay, Western Blot, Transfection, SDS Page, Mutagenesis, Luciferase, Reverse Transcription, Purification

Journal: Retrovirology

Article Title: The NTD-CTD intersubunit interface plays a critical role in assembly and stabilization of the HIV-1 capsid

doi: 10.1186/1742-4690-10-29

Figure Lengend Snippet: Electron microscopy of NTD-CTD interface mutants. HeLa cells were transfected with R9 plasmids containing either WT or alanine mutations. 24 hr post transfection, the cells were fixed in buffer containing 2.5% glutaraldehyde. Following embedding and staining, ultrathin sections were then examined in a FEI Tecnai Spirit Twin transmission electron microscope. Scale bars represent 100 nm Arrows indicate particles with conical capsids.

Article Snippet: For analysis of viral DNA synthesis by qPCR, viruses were produced in 293T cells using the 293T transfection reagent (Mirus), according to the manufacturer’s protocol.

Techniques: Electron Microscopy, Transfection, Staining, Transmission Assay, Microscopy

Journal: bioRxiv

Article Title: Dysregulation of Acid Ceramidase-mediated Sphingolipid Metabolism Contributes to Tumor Progression in Tuberous Sclerosis Complex

doi: 10.1101/2022.09.25.509382

Figure Lengend Snippet: 621-101 (TSC2-) and 621-103 (TSC2+) cells were treated with an ASAH1 inhibitor 17a (a) or carmofur (b) with indicated concentrations for 72 hr. Cell viability was measured using MTT assay (n=6/treatment group). (c) TSC2-null 621-101 cells were transfected with three independent ASAH1-siRNAs or control-siRNA for 48 hr. siRNA knockdown efficiency was determined by RT-PCR (n=3/group). (d) Cells were treated with ceramide, and then stained with Annexin V: FITC Apoptosis Detection Kit (BD#556547). Cell death was analyzed by flow cytometry (n=3). (e) The percentage of apoptotic (Annexin V+) cells was determined (percentage of Annexin V: FITC-positive cells in total cell number). (f) Cell viability was assessed 48 hr post siASAH1 RNA transfection in 621-101 cells using MTT assay. (g) siRNA knockdown efficiency was determined by immunoblotting. β-actin was used as a loading control. (h) 621-101 cells were infected with lentiviruses containing shRNA for vector pLKO, or ASAH1 , and then selected with puromycin for two weeks. Stable cells were harvested, and then stained with Annexin V: FITC Apoptosis Detection Kit. Cell death was analyzed by flow cytometry (n=3). (i) The percentage of apoptotic (Annexin V+) cells was determined (percentage of Annexin V: FITC-positive cells in total cell number). (j) Cell death was measured using PI exclusion assay. Relative cell death was compared between pLOK.1 and shRNA- ASAH1 cells. (k) Cell viability was measured using MTT assay (n=8-16/treatment group). *p<0.05; **p<0.01, Student t-test.

Article Snippet: 293T packaging cells were transfected with ASAH1 shRNA, or non-Targeting shRNA vectors using Mirus Trans-IT TKO Transfection reagent (Mirus).

Techniques: MTT Assay, Transfection, Control, Knockdown, Reverse Transcription Polymerase Chain Reaction, Staining, Flow Cytometry, Western Blot, Infection, shRNA, Plasmid Preparation, Exclusion Assay