Journal: bioRxiv

Article Title: DAMP-inducing Peptide Nanofibers and PAMP Combination Adjuvants Boost Functional Lung Tissue-resident Memory CD4 + T Cell Responses

doi: 10.1101/2024.08.28.610131

Figure Lengend Snippet: Lungs were collected from mice 4h and 24h after pulmonary administration of Cy5 + labeled KFE8-Ag85B nanofibers. The lung tissue was processed for flow cytometric analysis Following a hierarchal gating strategy, single cells were selected through doublet exclusion, followed by the selection of viable cells based on exclusion of cells that positively stained for viability dye. Then Cy5 + were selected as NF + cells. The expression of CD11b, CD11c, and MMR (CD206) was used to putatively identify interstitial macrophages (CD11b + CD11c + ), alveolar macrophages (CD11c + CD11b - MMR + ), and DCs (CD11c + CD11b - MMR - ) ( A ). The frequency of NF + cells is represented as a percentage of the total viable cell population in disrupted lung tissue 4h and 24h after i.t. delivery ( B ). Interstitial macrophages, alveolar macrophages, and DCs were analyzed for their expression of CD80 ( C-E ) and CD86 ( F-H ), which is represented as the MFI in untreated animals and 4 and 24 hours after treatment. Significant differences between treatment groups were determined by one-way ANOVA followed by a Tukey test to control for multiple comparisons (n = 4). *p<0.05.

Article Snippet: Cells were washed two times in permeabilization buffer and centrifuged 500 ×g for 5 minutes, and resuspended in an antibody cocktail containing anti-RORγt (Alexa Fluor 647, BD Biosciences, 562682), anti-T-bet (Brilliant Violet 785, Biolegend, 644835), anti-GATA3 (Brilliant Violet 711, BD Biosciences, 565449), and anti-EOMES (PE-Cy5, Thermo Fisher Scientific, 15-4875-82) antibodies to stain at room temperature for 30 minutes.

Techniques: Labeling, Selection, Staining, Expressing, Control

Journal: bioRxiv

Article Title: DAMP-inducing Peptide Nanofibers and PAMP Combination Adjuvants Boost Functional Lung Tissue-resident Memory CD4 + T Cell Responses

doi: 10.1101/2024.08.28.610131

Figure Lengend Snippet: Cy5-labeled peptide nanofibers were delivered via the pulmonary route and at indicated time points, Cy5 + cells were selected and identified through expression of surface markers described in Fig. S8. The percentage of identified APCs positive for Cy5 fluoresence are shown at 4h and 24h after treatment (A) . Each APC population was analyzed for the proportion of cells in that category that were Cy5 + (B) . Significant differences over time were determined by multiple t tests followed by a correction for multiple comparisons using the Holm-Sidak method (n = 4). *p<0.05, *p<0.005, ****p<0.0005.

Article Snippet: Cells were washed two times in permeabilization buffer and centrifuged 500 ×g for 5 minutes, and resuspended in an antibody cocktail containing anti-RORγt (Alexa Fluor 647, BD Biosciences, 562682), anti-T-bet (Brilliant Violet 785, Biolegend, 644835), anti-GATA3 (Brilliant Violet 711, BD Biosciences, 565449), and anti-EOMES (PE-Cy5, Thermo Fisher Scientific, 15-4875-82) antibodies to stain at room temperature for 30 minutes.

Techniques: Labeling, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic insights into complement pathway inhibition by CR1 domain duplication

doi: 10.1016/j.jbc.2024.107451

Figure Lengend Snippet: C3b and C4b cofactor activity of LHR-A domain variants. The indicated CR1 LHR-A domain variants ( Fig. 1 ) were tested in C4b ( A ) and C3b ( B ) cofactor activity assays. CSL040 containing LHR-ABC was used as a control/comparator cofactor activity is expressed as the % of intact C4b or C3b alpha-chain relative to un-cleaved C4b or C3b, respectively. Data points show the mean (±SD) C3b/C4b alpha chain (%) for each CR1 variant concentration (nM or pM) tested in combination with a constant Factor I concentration. IC 50 values calculated from these data are shown in Table 2 . CR1, complement receptor 1; LHR, long homologous repeat.

Article Snippet: For affinity in solution assays, a standard curve was generated using a 2-fold dilution series of plasma derived C3b or C4b (0.4875–125 nM; CompTech) prepared in running buffer and injected over the CR1-IgG4Fc captured surface.

Techniques: Activity Assay, Control, Variant Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic insights into complement pathway inhibition by CR1 domain duplication

doi: 10.1016/j.jbc.2024.107451

Figure Lengend Snippet: Potency of human soluble CR1 LHR-A domain variants in C3b and C4b cofactor activity assays

Article Snippet: For affinity in solution assays, a standard curve was generated using a 2-fold dilution series of plasma derived C3b or C4b (0.4875–125 nM; CompTech) prepared in running buffer and injected over the CR1-IgG4Fc captured surface.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic insights into complement pathway inhibition by CR1 domain duplication

doi: 10.1016/j.jbc.2024.107451

Figure Lengend Snippet: Affinity in-solution of CR1 and duplication variants. A , the method for generating affinity in solution data. On the left-hand side , CR1-IgG4Fc fusions of each CR1 variant/duplication to be tested were captured onto a protein G–coupled sensor surface, and then increasing concentrations of pdC3b or pdC4b added to generate standard response curves. As shown in the right-hand panel , affinity in-solution data was then generated by premixing increasing concentrations of untagged versions of each CR1 variant with 50 nM pdC3b or pdC4b and running each over their respective CR1-IgG4Fc captured protein G–coupled sensor surface to determine free C3b, using the previously generated standard curves as a reference. CR1 concentration was then plotted against free C3b ( B ) or free C4b ( C ). In both graphs, ligand concentration ( y -axis), measured as mean ± SD from N = 3 experiments each is plotted against total concentration of the respective CR1 truncation ( x -axis). See Table 6 for calculated dissociation constants derived from these graphs. CR1, complement receptor 1.

Article Snippet: For affinity in solution assays, a standard curve was generated using a 2-fold dilution series of plasma derived C3b or C4b (0.4875–125 nM; CompTech) prepared in running buffer and injected over the CR1-IgG4Fc captured surface.

Techniques: Variant Assay, Generated, Concentration Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Mechanistic insights into complement pathway inhibition by CR1 domain duplication

doi: 10.1016/j.jbc.2024.107451

Figure Lengend Snippet: Affinity in-solution dissociation constants of plasma-derived C3b and C4b to CR1 variants

Article Snippet: For affinity in solution assays, a standard curve was generated using a 2-fold dilution series of plasma derived C3b or C4b (0.4875–125 nM; CompTech) prepared in running buffer and injected over the CR1-IgG4Fc captured surface.

Techniques: Binding Assay

Journal: eLife

Article Title: Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

doi: 10.7554/eLife.92083

Figure Lengend Snippet: ( A ) Expression of E(spl)- m3, detected by single molecule fluorescent in situ hybridisation (smFISH), is lost upon triptolide treatment in Notch ON cells. Representative images show number of cytoplasmic RNA puncta in the volume sampled (n = 18, 16 for DMSO, triptolide) and intensity at the E(spl)-C (indicated by blue arrow for triptolide, n = 9, 8 for DMSO, triptolide). p-Values provided in . ( B ) Expression of E(spl)- m3, detected by smFISH, is lost upon A485 treatment in Notch ON cells. Representative images show the number of cytoplasmic RNA puncta in the volume sampled (n = 31, 22 for DMSO, A485) and intensity at the E(spl)-C (n = 13, 6 for DMSO, A485). ( C ) Representative images of H3K27Ac staining in Notch ON glands treated with DMSO and A485. H3K27Ac (top row) is lost throughout the nucleus and at E(spl)-C (blue arrows, middle row). Quantification of H3K27Ac levels at E(spl)-C (n = 19, 6 for DMSO and A485). p-Values provided in . ( D ) Average, profile, and max enrichment of Mam in Notch ON cells, treated with DMSO or Senexin A (n = 27, 26 for DMSO and Senexin). p-Values provided in . ( E ) Expression of E(spl)- m3, detected by smFISH, is lost in the Med13 KD in Notch ON cells compared to control KD (white RNAi). Representative images from the nucleus (centred on E(spl)-C, blue arrow) and quantification of intensity with boxplots, as described in (n = 10, 11 for control KD and Med13 KD). ( F ) Average, profile, and max enrichment of Hairless in Notch ON cells, treated with DMSO or Senexin B (n = 31, 28 for DMSO and Senexin B). All p-values provided in . ( G ) Average, profile, and max enrichment of CSL Hmut in Notch ON nuclei, treated with DMSO or Senexin B (n = 27, 31 for DMSO and Senexin B). All p-values provided in . ( H ) Average, profile, and max enrichment of Mam in Notch ON nuclei, treated with DMSO or NVP2 (n = 30, 43 for DMSO and NVP2). ( I ) Expression of E(spl- m3 ) , detected by smFISH, is lost upon NVP2 treatment in Notch ON cells. Representative images illustrate density of RNA puncta. (n = 31, 14 for DMSO and NVP2). All p-values provided in . ( J ) mRNA levels of nej (CBP/p300), Med13, and Cdk8 in tissues exposed to the respective RNAi or to control RNAi, measured by RT-qPCR and normalised to RPL32.

Article Snippet: For drug and hormonal treatments, dissected glands were incubated for an hour with the following compounds: triptolide (10 µM, Sigma-Aldrich T3652), ecdysone (5 µM, Cayman Chemicals 16145), A485 (5 µM, Cayman Chemicals 24119), Senexin A (1 µM, Tocris 4875), and Senexin B (2 µM, Cayman Chemicals 24119).

Techniques: Expressing, In Situ, Hybridization, Staining, Control, Quantitative RT-PCR

Journal: eLife

Article Title: Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

doi: 10.7554/eLife.92083

Figure Lengend Snippet:

Article Snippet: For drug and hormonal treatments, dissected glands were incubated for an hour with the following compounds: triptolide (10 µM, Sigma-Aldrich T3652), ecdysone (5 µM, Cayman Chemicals 16145), A485 (5 µM, Cayman Chemicals 24119), Senexin A (1 µM, Tocris 4875), and Senexin B (2 µM, Cayman Chemicals 24119).

Techniques: CRISPR, Modification, Software