Journal: Cell reports

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1016/j.celrep.2023.113478

Figure Lengend Snippet: (A) Representative brightfield images of A549ACE2 TP53ko pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h. Scale bars, 200 μm. (B) Top, western blotting analysis of whole cell lysates of A549ACE2 or A549ACE2 TP53ko pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h. Relative ratios of TP53/ACTB (normalized to EGFP -Dox) are annotated below TP53. Bottom, western blotting analysis of supernatants (SUP) of the corresponding cell constructs and treatments. (C) MSigDB pathway enrichment analysis of differential RNA-seq profiles upon doxycycline-induced expression (48 h, 1 μg/mL) of SARS-CoV-2 ancestral or Delta spike relative to expression of EGFP in either A549ACE2 or A549ACE2 TP53ko pInducer20 cells. n = 2 technical replicates/group. (D) Cell viability as assessed by CellTiter-Glo in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (Dox) for 72 h (mean ± SEM). The Welch two-sample t test with Holm p value correction was used to assess statistical significance: ***p < 0.001; **p < 0.01. n = 5 technical replicates/group. (E) RNA levels of CDKN1A by RT-qPCR in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (Dox) for 48 h (mean ± SEM). The Welch two-sample t test with Benjamini-Hochberg p value correction was used to assess statistical significance relative to the EGFP -Dox condition: ***p < 0.001; **p < 0.01; *p < 0.05; n.s., not significant. n = 4 technical replicates/group. (F) Senescence-associated beta-galactosidase activity in the indicated A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with or without 1 μg/mL doxycycline (Dox) for 48 h (mean ± SEM). The Welch two-sample t test with Holm p value correction was used to assess statistical significance: **p < 0.01; n.s., not significant. n = 14 technical replicates/group. (G) Left, ATAC-seq volcano plots of ancestral or Delta spike vs. EGFP expression in A549ACE2 pInducer20 cells 48 h after 1 μg/mL doxycycline induction. Right, number of significant (FDR < 0.05) ATAC-seq peaks upon doxycycline-induced expression (48 h, 1 μg/mL) of SARS-CoV-2 ancestral or Delta spike relative to expression of EGFP in either A549ACE2 or A549ACE2 TP53ko pInducer20 cells. Black triangles, peaks with increasing accessibility. Red circles, peaks with decreasing accessibility. n = 2 technical replicates/group. (H) Left, heatmap of relative levels of differentially expressed microRNAs (FDR < 0.05 and base mean > 10) from ancestral or Delta spike vs. EGFP expression in A549ACE2 or A549ACE2 TP53ko pInducer20 cells 48 h after 1 μg/mL doxycycline induction. Right, number of significant (FDR < 0.05) differentially expressed microRNAs upon doxycycline-induced expression (48 h, 1 μg/mL) of ancestral or Delta spike vs. EGFP expression in A549ACE2 or A549ACE2 TP53ko pInducer20 cells. Black triangles, significantly upregulated microRNAs. Red circles, significantly downregulated microRNAs. n = 2 technical replicates/group. (I) Heatmap of relative levels of cytokines in supernatants of A549ACE2 or A549ACE2 TP53ko pInducer20 cells treated with 1 μg/mL doxycycline for 48 h. The Welch two-sample t test with Benjamini-Hochberg p value correction was used to assess statistical significance. Sig, FDR < 0.05. n = 4 technical replicates/group. (J) Representative brightfield images of HNEpC (human nasal epithelial cell)-ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h. Scale bars, 200 μm. (K) Western blotting analysis of supernatants of HNEpC-ACE2 pInducer20 cells transduced with the indicated gene constructs and treated with or without 1 μg/mL doxycycline (Dox) for 48 h.

Article Snippet: After clarifying the supernatant, 50 μL 2x senescence reaction buffer (CST #78494) containing SA-beta-Gal substrate (CST #45954) and 10 mM beta-mercaptoethanol was added to 50 μL lysate and incubated at 37°C for 2 h. 50 μL of the reaction mix was then added to 50 μL 2x senescence stop solution, and fluorescence was read with excitation at 360 nm and emission at 465 nm with a SpectraMax iD3 plate reader.

Techniques: Transduction, Construct, Western Blot, RNA Sequencing Assay, Expressing, Quantitative RT-PCR, Activity Assay

Journal: Cell reports

Article Title: Differences in syncytia formation by SARS-CoV-2 variants modify host chromatin accessibility and cellular senescence via TP53

doi: 10.1016/j.celrep.2023.113478

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After clarifying the supernatant, 50 μL 2x senescence reaction buffer (CST #78494) containing SA-beta-Gal substrate (CST #45954) and 10 mM beta-mercaptoethanol was added to 50 μL lysate and incubated at 37°C for 2 h. 50 μL of the reaction mix was then added to 50 μL 2x senescence stop solution, and fluorescence was read with excitation at 360 nm and emission at 465 nm with a SpectraMax iD3 plate reader.

Techniques: FLAG-tag, Luciferase, Virus, Recombinant, SYBR Green Assay, Activity Assay, Software, CRISPR