Journal: bioRxiv

Article Title: Endogenous antigen processing promotes mRNA vaccine CD4 + T cell responses

doi: 10.1101/2025.03.11.642674

Figure Lengend Snippet: a , Schematic of mRNA-miR vaccine constructs encoding influenza NP or HA, followed by miR-targeting sequences in series. b , Schematic of expected antigen expression patterns and relevant antigen processing pathways for each mRNA-miR construct. c , Representative histograms, gated on live singlets, and d , summary data, of NP expression by flow cytometry after in vitro transfection with NP-miR mRNA. e-g , NP expression in murine lymph nodes after i.m. immunization with mRNA-LNPs. e , Representative composite images (top row) and magnified insets (bottom row) of inguinal lymph nodes. ER-TR7 is a fibroblastic reticular cell marker. Scale bars indicate 100 μm. f , Surface area of positive NP staining, relative to the entire lymph node surface area. g , Integrated intensity of NP staining among all NP + pixels. f,g , Bars indicate mean of 3 biological replicates +SEM. Each symbol represents one mouse. Analyzed by 1-way ANOVA; results of Dunnett’s multiple comparison test are shown. h , Representative western blot after transfection of C2C12 myotubes with NP-miR mRNA. i , Summary data of NP expression as in h , quantified relative to GAPDH using densitometry analysis. d,i , Symbols represent n=4-8 (d) or n=10 (i) biological replicates per condition, pooled from 3 (d) or 4 (i) independent experiments and normalized to the average %NP + value (d) or average NP/GAPDH value (i) among NP-control-transfected cells, per cell type within each experiment. Analyzed by linear mixed-effects model with cell type (d) and mRNA construct (d,i) treated as fixed effects and experiment treated as a random effect (d,i) . Significance of Dunnett’s multiple comparison test within each cell type is shown. c,h , PR8 influenza-infected and untreated controls are included for comparison.

Article Snippet: Cryosections were taken at 16 µm (iLN) or 10 µm (muscle), dried, and stored at -80°C until staining. iLN sections were permeabilized in 0.5% Triton X-100 for 15 minutes, blocked in 5% goat serum in PBS for 30 minutes, and stained overnight with Influenza A NP monoclonal antibody (Clone D67J) conjugated to FITC (Invitrogen MA1-7332, diluted 1:100) and Fibroblast Marker antibody (ER-TR7) conjugated to AF546 (Santa Cruz Biotechnology, sc-73355, diluted 1:50).

Techniques: Construct, Expressing, Flow Cytometry, In Vitro, Transfection, Marker, Staining, Comparison, Western Blot, Control, Infection

Journal: Advanced Healthcare Materials

Article Title: Mimicking the Dystrophic Cardiac Extracellular Environment through DystroGel

doi: 10.1002/adhm.202404251

Figure Lengend Snippet: Viability assessment. Representative confocal images and relative graphs of the live and dead assay on A) fibroblasts B) iPSC‐CMs C) iPSC‐SNs cultured for 15 days. The scale bars represent 100 µm. Student t‐ test was used to evaluate the differences between means; n = 3 sections per n = 3 biological replicates; significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the samples were incubated overnight at 4 °C with the following primary antibodies diluted 1:100: Vimentin (Sigma‐Aldrich, V5255); cardiac Troponin T (cTnT, Abcam, ab8295); β3‐Tubulin (β3TUBB, Biologend, 802 001), Fibroblasts marker (ER‐TR7, Santa Cruz Biotechnology, sc‐73355) Thereafter, the bulks were exposed to appropriate secondary antibodies (ThermoFisher, Waltham, MA, USA) for 2 h at RT.

Techniques: Cell Culture

Journal: Advanced Healthcare Materials

Article Title: Mimicking the Dystrophic Cardiac Extracellular Environment through DystroGel

doi: 10.1002/adhm.202404251

Figure Lengend Snippet: Fibroblasts encapsulation in dECM WT and DystroGel. A) Z‐stack images’ reconstruction of the whole WT (upper panel) and DystroGel (lower panel) samples and higher magnification views of two distinct regions of the specimen. The scale bars represent 500 and 200 µm, respectively. Volumetric 3D reconstruction of the samples at higher magnifications. Fibroblasts were stained with Vimentin (red), and the nuclei were detected with Hoechst (blue). B) Representative confocal images of fibroblasts in dECM WT and DystroGel. The cells were stained against Vimentin (red). Nuclei were counterstained with Hoechst (blue). Scale bars represent 50 µm; C) Quantitative RT‐PCR for the expression of TGF‐b, a‐SMA, HSPG2, and COL1A1 genes in fibroblasts (Fibro). Student t ‐test was used to evaluate the differences between means; n = 3; significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the samples were incubated overnight at 4 °C with the following primary antibodies diluted 1:100: Vimentin (Sigma‐Aldrich, V5255); cardiac Troponin T (cTnT, Abcam, ab8295); β3‐Tubulin (β3TUBB, Biologend, 802 001), Fibroblasts marker (ER‐TR7, Santa Cruz Biotechnology, sc‐73355) Thereafter, the bulks were exposed to appropriate secondary antibodies (ThermoFisher, Waltham, MA, USA) for 2 h at RT.

Techniques: Encapsulation, Staining, Quantitative RT-PCR, Expressing

Journal: Advanced Healthcare Materials

Article Title: Mimicking the Dystrophic Cardiac Extracellular Environment through DystroGel

doi: 10.1002/adhm.202404251

Figure Lengend Snippet: Multicellular 3D Model. A) Z‐stack image reconstruction of the whole WT (upper panel) and DystroGel (lower panel) samples. The scale bars represent 50 µm, respectively. ER‐TR7 (green), cTnT (red), and b3TUBB antibodies were used to stain fibroblasts, iPSC‐CMs, and iPSC‐SNs, respectively. Nuclei were counterstained with Hoechst (blue). B) Representative reconstruction of cellular organization within the 3D triculture model, created using Biorender. C) The graph indicates the area in the percentage of nerve endings labeled with b3TUBB inside dECM WT and DystroGel. Student t ‐test was used to evaluate the differences between means; n = 3 sections per n = 3 biological replicates; significant differences: * p < 0.05, ** p < 0.01, *** p < 0.001. D) Gene expression analysis related to iPSC‐SNs (NGF, TRKA, p75NTR, and MAP2), fibroblasts (TGF‐b, a‐SMA, and HSPG2) and iPSC‐CMs (NKX 2.5, NPPA and NPPB). Student t ‐test was used to evaluate the differences between means; n = 3; significant differences * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Then, the samples were incubated overnight at 4 °C with the following primary antibodies diluted 1:100: Vimentin (Sigma‐Aldrich, V5255); cardiac Troponin T (cTnT, Abcam, ab8295); β3‐Tubulin (β3TUBB, Biologend, 802 001), Fibroblasts marker (ER‐TR7, Santa Cruz Biotechnology, sc‐73355) Thereafter, the bulks were exposed to appropriate secondary antibodies (ThermoFisher, Waltham, MA, USA) for 2 h at RT.

Techniques: Staining, Labeling, Gene Expression