crl 1582 sup t1 cell line atcc  (ATCC)

Journal: Science Advances

Article Title: SLFN14 functions as a P-TEFb inhibitor to modulate the transcription of HIV-1 and cellular genes

doi: 10.1126/sciadv.adt7982

Figure Lengend Snippet: ( A ) Levels of infectious HIV-1and SIV viral particles produced in HEK293T cells transfected with SLFN14-Flag expressing vector and viral DNA constructs, as determined by infection of TZM-bl cells and quantification of firefly luciferase activity (top). Expression of SLFN14 was determined by Western blot (bottom). ( B ) Levels of infectious wild-type HIV-1 NL4-3 viruses from HEK293T cells transfected with viral DNA and various amounts of SLFN14-Flag expressing vector. Levels of SLFN14 were determined by Western blot (bottom). ( C ) The infectivity of VSV-G–pseudotyped HIV-1 NL4-3-E-R- in HEK293T cells transfected with SLFN14 -expressing vector determined by luciferase activity in infected cells. Expression of SLFN14 protein was determined by Western blot (bottom). ( D ) Quantification of the abundance of VSV-G–pseudotyped HIV-1 NL4-3-E-R— luc produced by HEK293T cells transfected with either 0.5 μg of shSLFN14 or green fluorescent protein-specific shRNA (shGFP), as determined by infection of SupT1 cells and quantification of firefly luciferase activity (top). The level of endogenous SLFN14 was determined by Western blot (bottom). ( E and F ) Flow cytometry analysis showing the effect of transiently expressed SLFN14-Flag protein on infection in both PBMCs (E) and Jurkat (F) by VSV-G–pseudotyped HIV-1 NL4-3-E-R- . The relative infection rate was calculated from the results of three experiments. ( G ) The infectivity of VSV-G–pseudotyped HIV-1 NL4-3-E-R— luc in Jurkat cells transfected with shSLFN14 or control shGFP vectors determined by luciferase activity in infected cells (top). The level of endogenous SLFN14 was determined by Western blot (bottom). ( H ) Flow cytometry analysis of p24 positive cell frequency in SLFN14 knockout Jurkat cells. The gRNA1, gRNA2, and gRNA3 represent three gRNAs targeting different sites of SLFN14 , respectively. Quantitative data are presented as means ± SD of three independent experiments with statistical significance (n.s., not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001) calculated using Student’s t test.

Article Snippet: SupT1 cells , ATCC , Catalog no. CRL-1942.

Techniques: Produced, Transfection, Expressing, Plasmid Preparation, Construct, Infection, Luciferase, Activity Assay, Western Blot, shRNA, Flow Cytometry, Control, Knock-Out

Journal: Science Advances

Article Title: SLFN14 functions as a P-TEFb inhibitor to modulate the transcription of HIV-1 and cellular genes

doi: 10.1126/sciadv.adt7982

Figure Lengend Snippet: ( A to D ) HEK293T cells were cotransfected with pNL4-3-Luc.R-E- , the VSV-G expression vector, and increasing levels of the SLFN14 -expressing vector. Levels of viral proteins in transfected HEK293T cells were determined by Western blot (A). Gag mRNA was quantified in HEK293T cells by RT-qPCR and normalized with glyceraldehyde-3-phosphate dehydrogenase (gapdh) mRNA levels (B). HIV-1 p24 levels were measured in supernatants by enzyme-linked immunosorbent assay (C). Infectious viruses in an equal volume of culture supernatant were quantified by measuring luciferase activity in infected SupT1 cells at 48 hours postinfection (D). ( E ) Levels of different viral RNA were determined by RT-qPCR in HEK293T cells cotransfected with pNL4-3-Luc.R-E- and VSV-G , and SLFN14 expression vectors (right). Levels of SLFN14 and Gag proteins were determined by Western blot (left). ( F ) Domains of SLFN14 and truncated regions of mutants. ( G to I ) HEK293T cells were cotransfected with pNL4-3-Luc.R-E- and expression vectors of VSV-G and wild-type SLFN14 or mutated versions. Expression of HIV-1 Gag and SLFN14 was analyzed by Western blot (G). Gag mRNA was quantified in HEK293T cells by RT-qPCR (H). Levels of progeny viruses were determined by infecting SupT1 cells and subsequent quantification of luciferase activity (I). Quantitative data are presented as means ± SD of three independent experiments with statistical significance (n.s., not significant; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001) calculated using Student’s t test.

Article Snippet: SupT1 cells , ATCC , Catalog no. CRL-1942.

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Infection