Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

doi: 10.1073/pnas.1701872114

Figure Lengend Snippet: Deletion of the NR0B1 microsatellite reduces NR0B1 expression, impairs A673 cell growth, and inhibits colony formation. (A) Sequencing results validating knockout of the NR0B1 GGAA-microsatellite about 1.5 kb upstream of the NR0B1 TSS in A673 cells. The sgRNAs targeted to either side of this region are underlined. GGAA-microsatellite is highlighted red, and CRISPR/Cas9 deleted region is highlighted blue. Gel shows deletion of NR0B1 microsatellite region compared with control (nondeleted), with densitometry quantification on Right (P < 0.01). Data are represented as mean ± SEM (n = 2). (B) NR0B1 mRNA (P < 0.05) and protein expression levels in control and CRISPR/Cas9-mediated knockout of NR0B1 microsatellite in A673 Ewing sarcoma cells, with Western blot densitometry quantification on Right. Control CRISPR/Cas9 plasmids do not contain sgRNAs. Data are represented as mean ± SEM (n = 3). (C) Growth and colony formation assay quantification of CRISPR/Cas9 control vs. NR0B1 microsatellite knockout in A673 cells (P < 0.05). Data are represented as mean ± SEM (n = 3).

Article Snippet: Mammalian expression constructs included the following: Lentiviral vectors containing CRISPR/Cas9 cDNA and sgRNA ( SI Materials and Methods ); retroviral vectors encoding Luc-RNAi and EF-2–RNAi and cDNAs for EWS/FLI, Δ22, R2L2, Mut9, and NR0B1 are previously described ( 4 , 13 , 22 , 23 ); the Mut9/R2L2 construct was ordered as a gene block (IDT) and cloned into the pMSCV hygro vector between EcoRI and HindIII restriction sites.

Techniques: Expressing, Sequencing, Knock-Out, CRISPR, Western Blot, Colony Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Role for the EWS domain of EWS/FLI in binding GGAA-microsatellites required for Ewing sarcoma anchorage independent growth

doi: 10.1073/pnas.1701872114

Figure Lengend Snippet: Deletion of the NR0B1 microsatellite in other cell lines. (A) NR0B1 mRNA and protein expression levels in control and CRISPR/Cas9-mediated knockout of the NR0B1 microsatellite in TC-71 and EWS/502 Ewing sarcoma cells (P < 0.05). Data are represented as mean ± SEM (n = 3). (B) Growth curves and soft agar assay quantification for NR0B1 microsatellite deletion in two other Ewing sarcoma cell lines (TC-71 cells and EWS/502 cells). Growth curve data are represented as mean ± SEM (n = 4). Control vs. CRISPR for TC-71 and EWS/502 cells are each statistically significant (P < 0.05). Soft agar data are represented as mean ± SEM (n = 2). (C) Densitometry quantification of PCR-amplified NR0B1-microsatellite–containing region for A673 control (wild-type NR0B1-microsatellite) allele vs. CRISPR-Cas9 knockout (deleted NR0B1-microsatellite) allele at different time points for up to 3 wk postlentiviral infection. (D) NR0B1 mRNA and protein expression levels in control and CRISPR/Cas9-mediated knockout of the NR0B1 microsatellite in non-Ewing sarcoma HEK293 cells. Data are represented as mean ± SEM (n = 3). n.s., not statistically significant. (E) NR0B1 protein levels and colony formation assay quantification for A673 cells with NR0B1 cDNA rescue in CRISPR/Cas9 control vs. microsatellite knockout. n.s., not statistically significant. Data are represented as mean ± SEM (n = 2).

Article Snippet: Mammalian expression constructs included the following: Lentiviral vectors containing CRISPR/Cas9 cDNA and sgRNA ( SI Materials and Methods ); retroviral vectors encoding Luc-RNAi and EF-2–RNAi and cDNAs for EWS/FLI, Δ22, R2L2, Mut9, and NR0B1 are previously described ( 4 , 13 , 22 , 23 ); the Mut9/R2L2 construct was ordered as a gene block (IDT) and cloned into the pMSCV hygro vector between EcoRI and HindIII restriction sites.

Techniques: Expressing, CRISPR, Knock-Out, Soft Agar Assay, Amplification, Infection, Colony Assay

Journal: PLoS Pathogens

Article Title: Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

doi: 10.1371/journal.ppat.1003106

Figure Lengend Snippet: Mature ( A ), germline ( B ), chimera 1 ( C ), or chimera 2 ( D ), IgGs were immobilized to anti-human IgG FC capture BLI biosensors and Env proteins were in solution at 1.2 µM. The QH0692 and D368R Envs were in gp120 form. The remaining Envs were in gp140 form (either as trimers or as monomers).

Article Snippet: The constant IgG splice variant cDNA (containing the two exons which encode the transmembrane and cytoplamic domains of human IgG) was synthesized by Genscript (Piscataway, NJ).

Techniques:

Journal: PLoS Pathogens

Article Title: Recombinant HIV Envelope Proteins Fail to Engage Germline Versions of Anti-CD4bs bNAbs

doi: 10.1371/journal.ppat.1003106

Figure Lengend Snippet: ( A ) Cell-surface expression of b12 mature, germline and chimeric BCRs on the surface of B cells. ( B ) Intracellular Ca 2+ flux mediated by the mature, germline and chimeric BCRs following BCRs cross-linking by goat anti-human IgG (H+L) F(ab′) 2 in A20 cells. ( C ) Binding of mature, germline and chimeras to SF162 gp140 trimer. ( D ) Intracellular Ca 2+ flux upon addition of SF162 gp140 trimers to B cells (DG-75) expressing the indicated b12 BCRs. ( E ) Binding of mature, germline and chimeras to QH0692 gp140 trimer. ( F ) Intracellular Ca 2+ flux upon addition of QH0692 gp140 trimers to B cells expressing the indicated b12 BCRs.

Article Snippet: The constant IgG splice variant cDNA (containing the two exons which encode the transmembrane and cytoplamic domains of human IgG) was synthesized by Genscript (Piscataway, NJ).

Techniques: Expressing, Binding Assay