gfp ahph  (Addgene inc)

Journal: Cell Insight

Article Title: TNF inhibits SARS-CoV-2 induced cell-cell fusion through activating the SDC4-RhoA signaling to promote actin bundles formation

doi: 10.1016/j.cellin.2026.100310

Figure Lengend Snippet: TNF inhibits SARS-CoV-2 spike induced cell-cell fusion by inducing actin bundles formation at cell-cell junctions via the RhoA/ROCK signaling pathway. (A) Representative confocal images of GFP-AHPH in sgcontrol, sgTRADD/TRAF2/RIPK1 complex, and sgSDC4 HEK293T cells treated with 10 ng/mL TNF for 30 min. Images are representative of five independent experiments. Scale bars represent 10 μm. (B) Quantification of GFP-AHPH fluorescence intensity. Data points represent the mean ± SEM from five independent experiments, with P values shown in figure. (C) Quantification of luciferase luminescence showing the effect of ROCK inhibitor Y27632 and TNF treatment on spike induced cell-cell fusion in HEK293T cells. Data points represent the mean ± SEM from four independent experiments, with P values shown in figure. (D) Immunoblot analysis showing the effect of ROCK inhibitor Y27632 and TNF treatment on S2′ cleavage in spike induced HEK293T syncytia. Blots are representative of three independent experiments. (E) Representative confocal images of GFP-AHPH in syncytia formed by HEK293T-S-HA and HEK293T-ACE2-V5 cells treated with PBS or TNF for 16 h. Green indicates AHPH, red indicates Spike-expressing cells, and white arrows indicate regions of AHPH enrichment or disappearance. Images are representative of three independent experiments. Scale bars represent 10 μm. (F) White lines indicate SARS-CoV-2 spike protein induced cell-cell transmission and quantify with fluorescence intensity of GFP-AHPH in (E). (G) Representative confocal images of GFP-AHPH in syncytia formed by HEK293T-S-HA and HEK293T-ACE2-V5 cells transfected with Vector or SDC4 for 16 h. Green indicates AHPH, red indicates S-expressing cells, and white arrows indicate regions of AHPH enrichment or disappearance. Images are representative of three independent experiments. Scale bars represent 10 μm. (H) White lines indicate SARS-CoV-2 spike protein induced cell-cell transmission and quantify with fluorescence intensity of GFP-AHPH in (G). (I) Representative confocal images of F-actin stained with phalloidin-488 in syncytia formed by HEK293T-S-HA and HEK293T-ACE2-V5 cells treated or untreated with 10 ng/mL TNF for 16 h. Green indicates F-actin, red indicates Spike-expressing cells, magenta indicates ACE2-expressing cells, and white arrows indicate regions of F-actin enrichment or disappearance. Images are representative of three independent experiments. Scale bars represent 10 μm. (J) White lines indicate SARS-CoV-2 spike protein induced cell-cell transmission and quantify with fluorescence intensity of F-actin in (I). (K) Representative confocal images of F-actin stained with phalloidin-488 in syncytia formed by HEK293T-S-HA and HEK293T-ACE2-V5 cells transfected with Vector or SDC4 overexpression for 16 h. Green indicates F-actin, red indicates Spike-expressing cells, and white arrows indicate regions of F-actin enrichment or disappearance. Images are representative of three independent experiments. Scale bars represent 10 μm. (L) White lines indicate SARS-CoV-2 spike protein induced cell-cell transmission and quantify with fluorescence intensity of F-actin in (K). (M) Representative confocal images of F-actin stained with phalloidin-488 in syncytia formed by HEK293T-S-HA and HEK293T-ACE2-V5 cells treated with 10 ng/mL TNF or 40 μM Y27632 for 16 h. Green indicates F-actin, red indicates S-expressing cells, magenta indicates ACE2-expressing cells, and white arrows indicate regions of F-actin enrichment or disappearance. Images are representative of three independent experiments. Scale bars represent 10 μm. (N) White lines indicate SARS-CoV-2 spike protein induced cell-cell transmission and quantify with fluorescence intensity of F-actin in (M).

Article Snippet: GFP-AHPH (Addgene plasmid #71368; http://n2t.net/addgene:71368 ; RRID: Addgene_71368) was from Addgene ( ).

Techniques: Fluorescence, Luciferase, Western Blot, Expressing, Transmission Assay, Transfection, Plasmid Preparation, Staining, Over Expression

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , GFP-AHPH plasmid , Addgene , Cat#:71368, RRID: Addgene_71368 , N/A.

Techniques: Purification, In Vitro, In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay, Plasmid Preparation, Sequencing

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/elife.98593

Figure Lengend Snippet: Figure 5. Activation of RhoA/ROCK pathway prevents authentic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced cell-cell fusion via forming actin bundles. (A) Representative confocal images of GFP-AHPH localization with or without 1 ng/mL interleukin-1β (IL-1β) treatment in 0.5 multiplicity of infection (MOI) wild-type (WT) authentic SARS-CoV-2-infected HEK293T-ACE2 cells at 6 and 24 hr post-infection (hpi). Schematics with green dots in the white dashed line boxes representing GFP-AHPH, red cycles representing SARS-CoV-2-infected cells, and white cycles representing neighboring cells. White arrowheads indicate the localization of GFP-AHPH, scale bars, 10 μm. Images are representative of three independent

Article Snippet: DOI: https://doi.org/10.7554/eLife.98593 21 of 29 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant protein Recombinant human IL- 1RA Peprotech 200- 01RA 1000 ng/mL Recombinant protein Recombinant human IL- 6 Peprotech 200- 06 100 ng/mL Recombinant protein Recombinant human IL- 8 Peprotech 200- 08M 100 ng/mL Recombinant protein Recombinant mouse IL- 1RA BioLegend 769706 In vivo: 150 μg/kg Commercial assay or kit Human IL- 1β ELISA kit R&D Systems DY201 N/A Commercial assay or kit RhoA pull- down activation assay Biochem kit Cytoskeleton BK036- S N/A Recombinant DNA reagent pVAX1 SARS- CoV- 2 spike (Wild type) plasmid This paper GenBank: QHD43419.1 Homo sapiens codon- optimized, HA- tag at the C- terminal Recombinant DNA reagent pVAX1 SARS- CoV- 2 spike (Alpha) plasmid This paper N/A Truncated 19 amino acids at the Cterminal Recombinant DNA reagent pVAX1 SARS- CoV- 2 spike (Beta) plasmid This paper N/A Truncated 19 amino acids at the Cterminal Recombinant DNA reagent pVAX1 SARS- CoV- 2 spike (Delta) plasmid This paper N/A Truncated 19 amino acids at the Cterminal Recombinant DNA reagent pVAX1 SARS- CoV- 2 spike (Omicron) plasmid GeneScript N/A Truncated 19 amino acids at the Cterminal Recombinant DNA reagent pcDNA4.0 human ACE2 plasmid This paper N/A V5- tag at the C- terminal Recombinant DNA reagent GFP- AHPH plasmid Addgene Cat#:71368, RRID:Addgene_71368 N/A Recombinant DNA reagent pRK5myc RhoA L63 plasmid Addgene Cat#:15900, RRID:Addgene_15900 N/A Sequence- based reagent PCR primers This paper PCR primers See Supplementary file 1 for primers used in this study Sequence- based reagent sgRNA primers This paper sgRNA primers See Supplementary file 2 for sgRNA primers used in this study Continued

Techniques: Activation Assay, Infection