Journal: PLoS Pathogens

Article Title: Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

doi: 10.1371/journal.ppat.1006397

Figure Lengend Snippet: A , B . Western blotting analysis of immunoprecipitation of TLR4 or TLR-4 FLAG with EBOV GP (A) or EBOV VP40 (B). Representative data from one of two independent experiments. C. Confocal microscopy of HPIV3/ΔF-HN/EboGP bound to primary CD4 + T lymphocytes, Jurkat cells and 293T-TLR4 cells. Insets show the formation of plasma membrane associated GP-positive puncti. D. Flow cytometry analysis demonstrating the binding of EBOV and HPIV3/ΔF-HN/EboGP to SupT1 T cells. E. Flow cytometry analysis of TLR4 expression by isolated CD4 + T cells. T cells were activated with CD3/CD28 beads and then cultured with EBOV. Results are representative of 3 donors. F . Inhibition of EBOV binding to SupT1 cells by anti-TLR4 serum: % of no serum control. Mean values ±SE based on triplicate samples of one of two independent experiments, *** P<0.001 (Student T-test).

Article Snippet: GP-TLR4 binding was determined by co-transfecting 293T cells with mammalian codon-optimized plasmids encoding EBOV GP or VP40, as well as the plasmids expressing TLR4 (Addgene, #20863) or TLR4 FLAG-tagged protein (TLR4-FLAG) (Addgene, #42646) using TransIT-LT1 reagent (Mirus) for 48 h at 37°C.

Techniques: Western Blot, Immunoprecipitation, Confocal Microscopy, Flow Cytometry, Binding Assay, Expressing, Isolation, Cell Culture, Inhibition

Journal: PLoS Pathogens

Article Title: Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

doi: 10.1371/journal.ppat.1006397

Figure Lengend Snippet: A, B. Western blot analysis of proteins involved in TLR4 signaling pathway TRAM1, p-TRAM1, MyD88, IRAK4, p-IRAK4, Pyk2, p-Pyk2, p38, p-p38 in SupT1 cells and monocytes (A) or THP-1, THP-1 MyD88-/- and SupT1 cells (B) following stimulations with LPS, poly I:C, HPIV3/EboGP, HPIV3/ΔF-HN/EboGP, HPIV3 or EBOV (A) or empty beads or EBOV GP beads (B) in the presence or absence of the TLR4 inhibitor CLI-095. C , D , E . Western blot analysis of p65 phosphorylation in SupT1 cells (C), monocytes (D) or THP-1, THP-1 MyD88-/- and SupT1 cells (E) following stimulation with CD3/CD28 beads, LPS (+, 100 ng/ml, ++; 500 ng/ml), VLP (+, 100 μl; ++, 250 μl) and HPIV3/ΔF-HN/EboGP (+, MOI 0.1 PFU/cell; ++, MOI 1 PFU/cell), empty beads or EBOV GP beads, as indicated, with or without CLI-095. Western blots in panels A-E are representative of two independent experiments. F . Percentages of dead (Live/Dead + ) cells, cell positive for caspase 8 and 9 and proliferated CD4 + T lymphocytes following a 4 day-long incubation with EBOV or LPS with or without CLI-095. Mean values ±SE based on triplicates from one of two independent experiments with P values * P <0.05, ** P <0.01, n.s., non-significant (Student T-test).

Article Snippet: GP-TLR4 binding was determined by co-transfecting 293T cells with mammalian codon-optimized plasmids encoding EBOV GP or VP40, as well as the plasmids expressing TLR4 (Addgene, #20863) or TLR4 FLAG-tagged protein (TLR4-FLAG) (Addgene, #42646) using TransIT-LT1 reagent (Mirus) for 48 h at 37°C.

Techniques: Western Blot, Incubation

Journal: bioRxiv

Article Title: CHD7 binds to insulators during neuronal differentiation

doi: 10.1101/2025.03.28.646031

Figure Lengend Snippet: (A-C) Fluorescence of mCherry and eYFP from inducible dual fluorescent reporter plasmid containing no sgRNA (empty sgRNA) and individual sgRNAs in dCas9-KRAB-MeCP2 iMOP cells undergoing early-stage neuronal differentiation (n=3, independent experiments). Box plots were used to compare mCherry levels between control and individual sgRNAs across different eYFP fluorescence bins. sgRNA1 didn’t show significant changes in mCherry level compared to empty sgRNA. sgRNA4 and sgRNA5 showed a significant decrease in mCherry level compared to the control empty sgRNA across different eYFP fluorescence bins. (D-F) Fluorescence of mCherry and eYFP from inducible dual fluorescent reporter plasmid containing no sgRNA (empty sgRNA) and individual sgRNAs in dCas9-p300-core cells undergoing early-stage neuronal differentiation (n=4, independent experiments). sgRNA1 didn’t show significant changes in mCherry level compared to empty sgRNA. sgRNA4 and sgRNA5 showed a significant decrease in mCherry level compared to the control empty sgRNA across different eYFP fluorescence bins. (G) Proposed model showing how CHD7 may function at an insulator to regulate gene expression. The Mir9-2 gene is located within the TAD boundary, where there are multiple CTCF+CHD7+ binding sites. Normally, CHD7 is enriched at the insulators to inhibit Mir9-2 gene expression. Disruption of the CTCF+CHD7+ sites by dCas9 complexes (CRISPRi and CRISPRa) increases Mir9-2 gene expression.

Article Snippet: Stable CRISPRi (dCas9-KRAB-MeCP2) cell line and CRISPRa (dCas9-p300-core) cell line were generated using lentiviral particles produced from either the Lenti_dCas9-KRAB-MeCP2 (Addgene #122205) or Lenti-EF1alpha-dCas9-p300_Blast (Addgene #192653).

Techniques: Fluorescence, Plasmid Preparation, Control, Gene Expression, Binding Assay, Disruption

Journal: bioRxiv

Article Title: CHD7 binds to insulators during neuronal differentiation

doi: 10.1101/2025.03.28.646031

Figure Lengend Snippet: Targeting CTCF+ CHD7+ regions around Mir9-2 (A-B) Fluorescence of mCherry and eYFP from inducible dual fluorescent reporter plasmid containing no sgRNA (empty sgRNA) and individual sgRNAs in dCas9-KRAB-MeCP2 iMOP cells undergoing early-stage neuronal differentiation (n=3, independent experiments). sgRNA2 and sgRNA3 did not show significant changes in mCherry level across different eYFP fluorescence bins compared to empty sgRNA. (C-D) Fluorescence of mCherry and eYFP from inducible dual fluorescent reporter plasmid containing no sgRNA (empty sgRNA) and individual sgRNAs in dCas9-p300-core cells undergoing early-stage neuronal differentiation (n=4, independent experiments). sgRNA2 and sgRNA3 showed a statistically significant decrease in mCherry level compared to the control empty sgRNA in some eYFP fluorescence bins.

Article Snippet: Stable CRISPRi (dCas9-KRAB-MeCP2) cell line and CRISPRa (dCas9-p300-core) cell line were generated using lentiviral particles produced from either the Lenti_dCas9-KRAB-MeCP2 (Addgene #122205) or Lenti-EF1alpha-dCas9-p300_Blast (Addgene #192653).

Techniques: Fluorescence, Plasmid Preparation, Control