Journal: Cell Communication and Signaling : CCS

Article Title: Senescent cells promote viral infection-associated inflammation and tissue damage through a robust NF-κB pathway

doi: 10.1186/s12964-025-02466-8

Figure Lengend Snippet: Senescent cells have a robust NF-κB pathway through target P65. A The RNA level of P65 was detected in the two groups by RT-PCR. B The protein level of P65 was detected in the two groups after CHX (10 µM) treatment by Western blot. C , D MG132 (20 µM, 4 h) and CQ (10 µM, 6 h) were added to the two groups of cells, respectively, to detect the protein expression level of P65 (C: natural senescent cells, D: DOX-induced senescent cells). E Two groups of cells were infected with HSV-1 and VSV, and the protein ubiquitination level was detected by Western blot. F The ubiquitination level of P65 was detected by Co-IP. G HEK293T cells were co-transfected with P65, PDLIM2, and Ub plasmids, and Co-IP was used to verify the regulatory effect of PDLIM2 on P65 ubiquitination. H , I ROS staining was performed to compare the two groups in PM and A549 cells, and their quantification plots were analyzed. J-M Two groups of cells were treated with NAC (5 mM) and H 2 O 2 , and ROS staining was performed ( J , K ). The relative expression levels of PDLIM2, IL-1β, and IL-6 were then detected by RT-PCR ( L , M )

Article Snippet: The pscAAV-PDLIM2-GFP overexpression system was constructed by inserting the PDLIM2 gene sequence into the pscAAV-GFP vector (Addgene, #32396).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Infection, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Transfection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Senescent cells promote viral infection-associated inflammation and tissue damage through a robust NF-κB pathway

doi: 10.1186/s12964-025-02466-8

Figure Lengend Snippet: Recovery of PDLIM2 alleviates virus infection-induced inflammation. A Two groups of cells were infected with lentivirus plenti-PDLIM2 to overexpress PDLIM2. After 48 h, cells were stimulated with HSV-1 and VSV, and the relative expression level of IL-1β was detected by RT-PCR. B Two groups of mice were infected with H1N1 for 7 days, and the expression of PDLIM2, P65, and p-P65 proteins in lung tissue was detected by Western blot. C HEK293T cells were infected with AAV-PDLIM2, and the expression of PDLIM2 was detected by RT-PCR. D , E AAV-PDLIM2 was injected into old mice (16–20 months old), followed by infection with HSV-1. The expression of PDLIM2 and the IL-1β level were then detected using RT-PCR. F-K AAV-PDLIM2 was injected into old mice (16–20 months old), then infected with HSV-1, and their representative H&E staining images and histological scores ( F-H ), along with representative F4/80 and IL-1β IHC images and their quantification plots ( I-K )

Article Snippet: The pscAAV-PDLIM2-GFP overexpression system was constructed by inserting the PDLIM2 gene sequence into the pscAAV-GFP vector (Addgene, #32396).

Techniques: Virus, Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Injection, Staining

Journal: bioRxiv

Article Title: Targeted and efficient AAV therapy for neuroblastoma via direct capsid-antibody coupling

doi: 10.1101/2025.11.12.687993

Figure Lengend Snippet: a. Schematic of the experimental protocol used for AAV-STITCH and mosaic capsid production by co-expressing plasmids encoding the AAV components together with the SpyCatcher fused to the anti-GD2 scFv (⍺GD2-SC). b . Western blot with anti-AAV antibody showing the capsid composition of SpyTag-modified and mosaic AAV preparations. c . On the right, western blot using an anti-V5 antibody to assess the coupling rate of the ⍺GD2-SC fusion to non-mosaic and mosaic (1:3 and 1:7; AAV9mut-ST/AAV9mut) AAV-STITCH capsids. On the left, densitometric quantification of the bands and calculation of the average number of ⍺GD2-SC molecules conjugated per viral capsid. n=3 independent biological replicates . d . On the left, immunofluorescence using anti-ZsGreen antibody on SH-SY5Y cells transduced with control AAV9mut-ST and ⍺GD2-SC (non-mosaic 1:0 and mosaic 1:3 - 1:7). Scale bar: 100 µm. On the right, bar graph showing the quantification of ZsGreen+ cells over total cells in the different experimental conditions. N = 3 independent biological replicates. e. RT-qPCR quantification of cell membrane–bound viral genomes (4°C assay) following incubation with control AAV9mut-ST alone or conjugated with ⍺GD2-SC (non-mosaic 1:0 and mosaic 1:3 - 1:7). n = 3 independent biological replicates. f . RT-qPCR quantification of viral genome copies in SH-SY5Y cells transduced with control AAV9mut-ST alone or conjugated with ⍺GD2-SC (non-mosaic 1:0 and mosaic 1:3 - 1:7) at 37°C. n = 3 independent biological replicates. Error bars represent SD. *p<0.05; **p<0.01; ***p < 0.001, One-way ANOVA with Tukey’s multiple comparisons correction.

Article Snippet: The SpyCatcher peptide sequence, fused to the scFv-anti-GD2 (a kind gift from M. Brenner), was synthesized by Azenta Genewiz and subcloned into pcDNA3.1 plasmid (Addgene) with a V5 tag inserted between the two peptides and a His-tag added at the C-terminus of the construct. scAAV-CMV vectors carrying the ZsGreen, Luciferase, and HSV-TK transgenes were generated by amplifying the respective coding sequences (CDS) and cloning them into the scAAV-GFP backbone (Addgene #32396) using BamHI and NotI restriction sites.

Techniques: Expressing, Western Blot, Modification, Immunofluorescence, Transduction, Control, Quantitative RT-PCR, Membrane, Incubation

Journal: bioRxiv

Article Title: Targeted and efficient AAV therapy for neuroblastoma via direct capsid-antibody coupling

doi: 10.1101/2025.11.12.687993

Figure Lengend Snippet: Representative anti-V5 Western blot showing the detection of SpyCatcher fused to the anti-GD2 scFv (⍺GD2-SC) alone or co-expressed with AAV9-mut-ST either post-production ( a ) or during viral production ( b ). c . Box plot showing the viral genome titers, assessed by RT-qPCR, from multiple independent productions of AAV9, AAV9mut, AAV9mut-ST, and 1:3 and 1:7 mosaic preparations (n > 3 experimental replicates per group). d . Left: Anti-AAV Western blot showing the capsid composition of AAV9mut-ST alone or conjugated to ⍺GD2-SC. Right: Densitometric quantification of the proportion of unconjugated VP3 (gray) and VP3 conjugated with ⍺GD2-SC (orange). e . On the left, immunofluorescence using anti-ZsGreen antibody on SH-SY5Y cells transduced with control AAV9mut-ST alone or couple with ⍺GD2. Scale bar: 100 µm. On the right, bar graph showing the quantification of ZsG+ cells over total cells in the different experimental conditions. n=3 independent biological replicates. Error bars represent SD. **p < 0.01; t-test.

Article Snippet: The SpyCatcher peptide sequence, fused to the scFv-anti-GD2 (a kind gift from M. Brenner), was synthesized by Azenta Genewiz and subcloned into pcDNA3.1 plasmid (Addgene) with a V5 tag inserted between the two peptides and a His-tag added at the C-terminus of the construct. scAAV-CMV vectors carrying the ZsGreen, Luciferase, and HSV-TK transgenes were generated by amplifying the respective coding sequences (CDS) and cloning them into the scAAV-GFP backbone (Addgene #32396) using BamHI and NotI restriction sites.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Transduction, Control