pcaggs expression vector  (Addgene inc)

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , Schematic representation of the constructs used for transfection and flow cytometry. In the VSV-G backbone with tGFP-P2A-BFP sequence, the SP and 62 AA of the mature G protein are exchanged by the corresponding signal peptide-like targeting sequence and 62 AA of the mature DENV subunit. The AA sequence of each targeting sequence is shown. Note that an extra methionine (black) is added at the N-terminus of each DENV targeting sequence for translation initiation. HEK293T cells were transiently transfected, treated with FT for 18 h, and analyzed by flow cytometry for tGFP and BFP expression levels. Graph shows the four parameter concentration-response curves of FT for the different constructs for tGFP expression (mean fluorescent intensity). Relative protein expression was calculated by the ratio tGFP:BFP, normalized to untreated transfection control for each construct (set at 1.0). Values are mean ± SD; n=3. Note that the two blue curves coincide. B , Same as in (A), but for constructs in which the SP only of G was exchanged by the corresponding signal peptide-like targeting sequences (C 14 , M 21 or E 23 ) of the different DENV subunits. Graph shows mean ± SD; n=3. C , Same as in (B) but for the C 14 signal peptide-like targeting sequences of the other serotypes of DENV and of ZIKV. The AA sequence of each C 14 SP is shown. Note that an extra methionine is added at the N-terminus of each flaviviral targeting sequence for translation initiation. Graph shows mean ± SD; n=3. The grey curve represents the VSV-G-tGFP-P2A-BFP control with the SP of WT VSV-G.

Article Snippet: For flow cytometry experiments, the VSV-G protein (from VSV-G expressing pMD2.G plasmid, Didier Trono Lab, Addgene) was inserted into a pCAGGS-tGFP-P2A-BFP expression vector after linearization of the vector via EcoRV cleavage (New England Biolabs, Ipswich, Massachusetts, USA).

Techniques: Construct, Transfection, Flow Cytometry, Sequencing, Expressing, Concentration Assay, Control

Journal: bioRxiv

Article Title: Sec61 translocon inhibitor Flavitransin blocks selectively dengue virus polyprotein insertion into the ER membrane with pan-flavivirus antiviral potency

doi: 10.1101/2025.03.29.646078

Figure Lengend Snippet: A , DENV2 was passaged on Huh7 cells under FT pressure. Compound concentration was gradually increased when viral cytopathic effect on cells was detected. After 75 passages, virus was cultivated in the absence of compounds to generate virus stocks for downstream analysis. At passage 50 and 75, virus was collected and genotyped by nanopore sequencing (see panel C). FT resistance was selected in 2 independent cell cultures. As controls, a culture with FT IN or with control medium was included that underwent the same passaging procedure. Cartoon created with BioRender (2025); www.bioRender.com . B , At end-point, virus stocks were evaluated in Huh7 cells for sensitivity to FT. A virus stock from a low passage (# 3; light blue) was included as reference. Cells were infected with the different virus stocks (MOI ∼ 0.5) and treated with increasing concentrations of FT. After 4-5 days post infection, virus yield in the supernatant was quantified by RT-qPCR detection of the viral 3’UTR. Graph shows the four parameter concentration-response curves of FT for virus release from infected cells. Values (mean ± SD, from two independent experiments; n=2) represent the viral load in FT-treated cells as normalized to the viral copy number of each untreated virus control. C , Summary of the detected AA mutations in the different viral clones as compared to the initial DENV2 control strain (# 3). The numbers in each colored box refer to the residues of the respective viral subunit. The mutations in the medium control (dark blue) and FT IN (grey) are shown at the top; the two FT-resistant viruses (dark and light red) are shown at the bottom. D , Schematic representation of the hydrophobic mutants of the DENV2 C 14 SPs. HEK293T cells were transiently transfected with the VSV-G-tGFP-P2A-BFP constructs and treated with FT for 18 h. GFP and BFP fluorescence (mean fluorescent intensity) was quantified by flow cytometry and relative tGFP:BFP expression ratio (normalized to untreated transfection control) was determined. Graph on the left shows the four parameter concentration response curves of FT for VSV expression for different C14 mutants from three independent experiments. Values are mean ± SD; n=3. Graph at the right shows the relative tGFP:BFP expression ratios at 2 µM FT for additional hydrophobic mutants (A102V/I/L/M/F/Y/W). Values are mean ± SD; n=3.

Article Snippet: For flow cytometry experiments, the VSV-G protein (from VSV-G expressing pMD2.G plasmid, Didier Trono Lab, Addgene) was inserted into a pCAGGS-tGFP-P2A-BFP expression vector after linearization of the vector via EcoRV cleavage (New England Biolabs, Ipswich, Massachusetts, USA).

Techniques: Concentration Assay, Virus, Nanopore Sequencing, Control, Passaging, Infection, Quantitative RT-PCR, Clone Assay, Transfection, Construct, Fluorescence, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Acute depletion of BRG1 reveals its primary function as an activator of transcription

doi: 10.1038/s41467-024-48911-z

Figure Lengend Snippet: a Western blotting shows efficient depletion of BRG1 by Auxin treatment for 6, 10, and 24 h. Primary CD4 + T cells were isolated from BRG1-AID mice or wild-type mice. Following transduction of the cells with osTIR1 expression viral particles for 24 h, the cells were treated with Auxin for the times indicated on the top (6, 10, and 24 h). WT indicates the cells isolated from wildtype B6 mice; AID indicates the cells isolated from Brg1-AID knock-in B6 mice. Beta-actin and histone H3 were used as protein loading controls (repeated ≥ 2 times independently with similar results; uncropped scans of Fig. 1a are supplied as Source Data files). b A genome browser snapshot showing the BRG1 ChIC-seq signals in wild-type cells and BRG1-depleted cells. The cells were treated with Auxin for 10 h as described in Panel ( a ). Blue tracks: two replicates for wild-type cells; Red tracks: two panels for BRG1-depleted cells. c MA plot illustrating the genome-wide reduction in BRG1 binding (data from Panel ( b ) following 10 h of BRG1 depletion. Significantly altered BRG1 peaks are indicated, with decreases in red and increases in blue, from a total of 29,986 peaks. Significance was determined using a two-sided ROTS statistical test, with a cutoff of P < 0.05 and fold-change > 1.3. The P-values were calculated using the two-sided ROTS method, optimizing the reproducibility of feature ranking. d Bar plot showing the number of the BRG1 peaks at TSS, enhancers, and other regions in the wild-type CD4 + T cells. Note that enhancers are defined by the H3K27ac ChIC-seq data. e Bar plot showing the fractions of decreased BRG1 peaks at TSS, enhancers, and other regions in CD4 + T cells after BRG1 depletion for 10 h.

Article Snippet: The osTIR1 expression vector was obtained from Addgene (Plasmid #86233) .

Techniques: Western Blot, Isolation, Transduction, Expressing, Knock-In, Genome Wide, Binding Assay