Journal:

Article Title: Inhibition of cellular proliferation by the Wilms tumor suppressor WT1 requires association with the inducible chaperone Hsp70

doi:

Figure Lengend Snippet: Transcriptional activation of hsp70 by WT1. (A) Induction of Hsp70 protein following expression of WT1. U2OS cells with inducible expression of WT1 were grown in the presence or absence (24 hr) of tetracycline, and equal amounts of cellular lysates were analyzed by immunoblotting with antibody against Hsp70. Induction of WT1 is shown at bottom. (B) Induction of hsp70.1 mRNA by WT1. Northern blot analysis of U2OS cells with inducible WT1, EWS–WT1, or empty vector, following growth in the presence or absence (24 hr) of tetracycline. A gene-specific probe was derived from the 3′ untranslated region of human hsp70.1. (Middle) Reprobing of the blot with a WT1 cDNA to confirm inducible expression of WT1 and the EWS–WT1 chimera; (bottom) a GAPDH loading control. (C) Transcriptional activation of the hsp70 promoter by WT1. U2OS cells were transfected with CMV-driven WT1(−KTS) or empty vector, along with reporter constructs, followed by determination of CAT activity. The respective fragments of the hsp70 promoter reporter are shown (bottom), including the primary HSE and CCAAT regulatory elements. The HSE–CAT reporter contains multimerized HSE sites. The fold induction of CAT activity was determined by scintillation counting.

Article Snippet: Transfection efficiency was standardized by cotransfection of a human growth hormone reporter (Nichols Institute), CAT activity was determined by use of standard procedures, and quantitated by scintillation counting, following chromatography on TLC plates.

Techniques: Activation Assay, Expressing, Western Blot, Northern Blot, Plasmid Preparation, Derivative Assay, Transfection, Construct, Activity Assay

Journal: Journal of Virology

Article Title: Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model

doi: 10.1128/JVI.01442-19

Figure Lengend Snippet: Characterization of guinea pig PDGFRA as the receptor for PC-independent GPCMV infection of fibroblast cells. (a) Western blot analysis for the detection of endogenous PDGFRA in various guinea pig cell types. Lanes (cell lysates): 1, renal epithelial (REPI) cells; 2, trophoblast epithelial (TEPI) cells; 3, fibroblasts (GPL); 4, loading dye-only control; 5, GPL fibroblasts plus transient expression plasmid for gpPDGFRA (GenScript). (b) β-Actin as a Western blotting control for cell lysate lane loading in panel a. (c) CRISPR/Cas9 knockout of PDGFRA from a guinea pig fibroblast cell line verified by Western blot analysis. Lanes (cell lysates): 1, GPL fibroblasts; 2, GPL fibroblasts expressing Cas9; 3, GPL PDGFRA knockout (GPKO) cells; 4, GPL fibroblasts plus transient gpPDGFRA expression plasmid. (d) β-Actin as Western blotting control for cell lysate lane loading for panel c. The arrows in panels a and c indicate the location of gpPDGFRA. (e) CRISPR guide RNA (gRNA) targets (boxed). CRISPR A gRNA targets bases 214 to 233; CRISPR C gRNA targets bases 149 to 168. The locus in both the wild type and the clonal mutant cell line was PCR cloned and sequenced. The alignment shows part of exon 2 with the deletion of nucleotides 153 to 217 in CRISPR-treated cells. The sequences of the wild type (wt_exon2_PDGFRA) and the knockout mutant (PCR_CRISPR_KO 1) are shown. The alignment was performed using MacVector software. (f) GPCMV PC− growth curve on GPL or GPKO fibroblasts. (g) GPCMV PC+ growth curve on GPL or GPKO fibroblasts. (h) HSV-1 infection on GPL and GPKO fibroblasts at 60 h postinfection. The MOI was 1 PFU/cell for growth curves (f to h).

Article Snippet: Lanes (cell lysates): 1, renal epithelial (REPI) cells; 2, trophoblast epithelial (TEPI) cells; 3, fibroblasts (GPL); 4, loading dye-only control; 5, GPL fibroblasts plus transient expression plasmid for gpPDGFRA (GenScript). (b) β-Actin as a Western blotting control for cell lysate lane loading in panel a. (c) CRISPR/Cas9 knockout of PDGFRA from a guinea pig fibroblast cell line verified by Western blot analysis.

Techniques: Infection, Western Blot, Control, Expressing, Plasmid Preparation, CRISPR, Knock-Out, Mutagenesis, Clone Assay, Software

Journal: Scientific Reports

Article Title: Discovery of SARS-CoV-2 antiviral synergy between remdesivir and approved drugs in human lung cells

doi: 10.1038/s41598-022-21034-5

Figure Lengend Snippet: FDA-approved compounds synergize with low dose remdesivir in primary human bronchial epithelial cells. (a) (b) RT-qPCR quantifying SARS-CoV-2 genome equivalents at 96hpi of Normal Human Bronchial Epithelial (NHBE) cells transiently expressing hACE2 treated with the indicated drug combinations and infected with SARS-CoV-2 at MOI of 5. Drug was added at 40 μM except remdesivir, which was added at ( a ) 0.37 μM or ( b ) 0.13 μM, and velpatasvir, which was added at 10 mM to maintain the ratio of 1:4 in dosing with its combination sofosbuvir. Data represent four combined replicates from three independent experiments and genome copy number was normalized to DMSO within the same experiment. R: remdesivir; V: velpatasvir; S: sofosbuvir; E: elbasvir; G: grazoprevir.

Article Snippet: Normal Human Bronchial Epithelial (NHBE) cells transiently expressing hACE2 were obtained from Lonza (Basel, Switzerland) (cat. no. CC-2540) and maintained in bronchial epithelial cell growth medium (Lonza, Basel, Switzerland; CC-3170).

Techniques: Quantitative RT-PCR, Expressing, Infection