Journal: Scientific Reports

Article Title: Deciphering the role of protein kinase A in the control of FoxP3 expression in regulatory T cells in health and autoimmunity

doi: 10.1038/s41598-024-68098-z

Figure Lengend Snippet: Low TCR stimulation activates PKA in human Tconv cells. ( a ) Scatter plots showing PKA activity in human mPKAI-Tconv and CTR-Tconv cells TCR-stimulated for 5 and 15 min. Data are shown from four independent experiments in duplicates (n = 8). ( b ) Left, immunoblot showing phosphorylated (p) and total CREB in human mPKAI-Tconv and CTR-Tconv cells TCR-stimulated or not for 15 min. Right, relative densitometric quantitation of p-CREB in the aforementioned experimental conditions. Data are shown from nine independent experiments (n = 9); uncropped blots are presented in Supplementary Fig. . ( c ) ChIP assay for CREB on FoxP3 promoter, CNS2 and CNS3 regions of mPKAI-Tconv and CTR-Tconv cells TCR-stimulated for 10 min. The horizontal line (bracket ± SEM) indicates the percent of input from a control ChIP (Ab:non-immune serum). Data are shown from three independent experiments in duplicates (n = 6). Independet experiments refer to different individuals.

Article Snippet: For the simultaneous evaluation of surface and intracellular molecules, human iTreg cells were stained with the following antibodies: FITC anti-CD4 (Clone RPA-T4, Cat: 561842) (BD Pharmingen), PE-Cy7 anti-CD25 (Clone M-A251, Cat: 560920) (cBD Pharmingen), APC anti-CD152/CTLA-4 (Clone BNI3, Cat: 555855) (BD Pharmingen), PE-Cy5 anti-GITR (Clone REA1007, Cat: 130-116-842) (Miltenyi Biotec), BV421 anti-CD279/PD-1 (Clone EH12.1, Cat: 562516) (BD Horizon), PE anti-FoxP3-All (Clone PCH101, Cat: 560046) (eBioscience) and PE anti-FoxP3-E2 (Clone 150D/E4, Cat: 12-4774-42) (BD Pharmingen).

Techniques: Activity Assay, Western Blot, Quantitation Assay, Control

Journal: Scientific Reports

Article Title: Deciphering the role of protein kinase A in the control of FoxP3 expression in regulatory T cells in health and autoimmunity

doi: 10.1038/s41598-024-68098-z

Figure Lengend Snippet: Inhibition of PKA impairs FoxP3 induction. ( a ) Scatter plots showing FoxP3-All (left) and FoxP3-E2 (right) mRNA levels in mPKAI-Tconv and CTR-Tconv cells TCR-stimulated or not for 24 or 36 h, respectively. Data are shown from four independent experiments in duplicates (n = 8). ( b ) Left, immunoblot analysis of FoxP3-All, FoxP3-E2 and ERK1/2 in mPKAI-Tconv and CTR-Tconv cells TCR-stimulated for 36 h. Right, relative densitometric quantitation of FoxP3-All or FoxP3-E2 normalized on ERK 1/2 in the aforementioned experimental conditions. Data are shown from thirteen independent experiments (n = 13). ( c ) Left, immunoblot analysis of p-STAT5 and p-S6 in human Tconv cells, as described in B. Right, relative densitometric analysis of p-STAT5 and p-S6 normalized on their total proteins, respectively. Data are shown from five independent experiments in duplicates (n = 10). Independet experiments refer to different individuals. All the uncropped blots are presented in Supplementary Fig. .

Article Snippet: For the simultaneous evaluation of surface and intracellular molecules, human iTreg cells were stained with the following antibodies: FITC anti-CD4 (Clone RPA-T4, Cat: 561842) (BD Pharmingen), PE-Cy7 anti-CD25 (Clone M-A251, Cat: 560920) (cBD Pharmingen), APC anti-CD152/CTLA-4 (Clone BNI3, Cat: 555855) (BD Pharmingen), PE-Cy5 anti-GITR (Clone REA1007, Cat: 130-116-842) (Miltenyi Biotec), BV421 anti-CD279/PD-1 (Clone EH12.1, Cat: 562516) (BD Horizon), PE anti-FoxP3-All (Clone PCH101, Cat: 560046) (eBioscience) and PE anti-FoxP3-E2 (Clone 150D/E4, Cat: 12-4774-42) (BD Pharmingen).

Techniques: Inhibition, Western Blot, Quantitation Assay

Journal: Scientific Reports

Article Title: Deciphering the role of protein kinase A in the control of FoxP3 expression in regulatory T cells in health and autoimmunity

doi: 10.1038/s41598-024-68098-z

Figure Lengend Snippet: PKA inhibition affects the immunometabolic asset and phenotype of Tconv cells during their diffentiation towards iTreg cells and their suppressive function ( a ) Left, kinetic profile of ECAR in human mPKAI-Tconv and CTR-Tconv cells TCR-stimulated or not for 12 h. ECAR was measured in real time, under basal conditions and in response to glucose, oligomycin, and 2-DG. Data are shown from three independent experiments at least in technical duplicates (n = 10). Right, parameters of the glycolytic pathway were calculated from the ECAR profile of Tconv cells in the above-mentioned conditions. Data are expressed as mean ± SEM of three different measurements, each of them in ten replicates (n = 30). ( b ) Representative dot plots (left) and cumulative data (right) of CD25, FoxP3-All and FoxP3-E2 in mPKAI-Tconv and CTR-Tconv cells. Data are shown from eleven independent experiments (n = 11). ( c ) Scatter plots showing the expression of CTLA-4, PD-1 and GITR gated on CD4 + FoxP3-All + (top) or CD4 + FoxP3-E2 + (bottom) in mPKAI-Tconv and CTR-Tconv cells. Data are shown as mean ± SEM from four independent experiments in duplicates (n = 8). ( d ) Left, flow cytometry histograms showing proliferation of CFSE + CD4 + T cells TCR-stimulated for 96 h in vitro and cultured alone (empty curves) or in the presence of various numbers of flow-sorted iTreg from mPKAI-Tconv and CTR-Tconv cells. Numbers in plots indicate the percent of CSFE dilution in CD4 + T cells cultured alone (top left) and co-cultured with iTreg cells (above bracketed lines), as indicated. Right, cumulative data of CD4 + T cell proliferation in the above conditions. Data are shown from four independent experiments in duplicates (n = 8). Independet experiments refer to different individuals.

Article Snippet: For the simultaneous evaluation of surface and intracellular molecules, human iTreg cells were stained with the following antibodies: FITC anti-CD4 (Clone RPA-T4, Cat: 561842) (BD Pharmingen), PE-Cy7 anti-CD25 (Clone M-A251, Cat: 560920) (cBD Pharmingen), APC anti-CD152/CTLA-4 (Clone BNI3, Cat: 555855) (BD Pharmingen), PE-Cy5 anti-GITR (Clone REA1007, Cat: 130-116-842) (Miltenyi Biotec), BV421 anti-CD279/PD-1 (Clone EH12.1, Cat: 562516) (BD Horizon), PE anti-FoxP3-All (Clone PCH101, Cat: 560046) (eBioscience) and PE anti-FoxP3-E2 (Clone 150D/E4, Cat: 12-4774-42) (BD Pharmingen).

Techniques: Inhibition, Expressing, Flow Cytometry, In Vitro, Cell Culture

Journal: Scientific Reports

Article Title: Deciphering the role of protein kinase A in the control of FoxP3 expression in regulatory T cells in health and autoimmunity

doi: 10.1038/s41598-024-68098-z

Figure Lengend Snippet: Reduced CREB phosphorylation associates with low FoxP3 expression in Tconv cells during their differentiation towards iTreg cells from autoimmune RR-MS subjects. ( a ) Left, immunoblot analysis of total and p-CREB in human Tconv cells from healthy and RR-MS subjects TCR-stimulated at different time points. Right, relative densitometric quantitation of p-CREB normalized on total CREB in the aforementioned experimental conditions. ( b ) Left, immunoblot analysis of FoxP3-All, FoxP3-E2 and ERK 1/2, in TCR-stimulated Tconv cells from healthy (n = 5) and RR-MS (n = 5) subjects. Right, relative densitometric quantitation of FoxP3-All and FoxP3-E2 normalized on ERK 1/2 in above conditions. Data are shown as mean ± SEM from five independent experiments (5 healthy and 5 RR-MS subjects) in triplicates (n = 15). All the uncropped blots are presented in Supplementary Fig. . ( c ) Statistical correlation between expression levels of basal p-CREB from ex-vivo Tconv cells with FoxP3-E2 in healthy (n = 5) and RR-MS (n = 5) subjects, in the above mentioned conditions. All the uncropped blots are presented in Supplementary Fig. .

Article Snippet: For the simultaneous evaluation of surface and intracellular molecules, human iTreg cells were stained with the following antibodies: FITC anti-CD4 (Clone RPA-T4, Cat: 561842) (BD Pharmingen), PE-Cy7 anti-CD25 (Clone M-A251, Cat: 560920) (cBD Pharmingen), APC anti-CD152/CTLA-4 (Clone BNI3, Cat: 555855) (BD Pharmingen), PE-Cy5 anti-GITR (Clone REA1007, Cat: 130-116-842) (Miltenyi Biotec), BV421 anti-CD279/PD-1 (Clone EH12.1, Cat: 562516) (BD Horizon), PE anti-FoxP3-All (Clone PCH101, Cat: 560046) (eBioscience) and PE anti-FoxP3-E2 (Clone 150D/E4, Cat: 12-4774-42) (BD Pharmingen).

Techniques: Expressing, Western Blot, Quantitation Assay, Ex Vivo

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A) Quantification of intracellular replicating (eFluor - ) and non-replicating (eFluor + ) E. faecalis strains from macrophage lysates at 2, 6, and 20 hpi using flow cytometry. WT OG1RF fixed with 4% PFA prior to infection (Fixed WT) was included as non-proliferating controls. Representative histograms from n = 2-4 are shown. (B) Proportion (%) of intracellular replicating E. faecalis (eFluor - ) population from WT and mutant E. faecalis -infected macrophage lysates. Bars represent mean ± SD of n = 4, except fixed WT (n = 2). Statistical significance of each strain against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. Only comparisons with p < 0.05 are annotated. ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. (C) Representative confocal microscopy images of RAW264.7 macrophages infected by E. faecalis strains pre-stained with eFluor 670 (magenta) at 20 hpi from n = 3. Samples were fixed and post-stained for Enterococcus -specific Group D antigen (green), double-stranded DNA (dsDNA; blue) and F-actin (white).

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Flow Cytometry, Infection, Mutagenesis, Confocal Microscopy, Staining

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A) LDH cytotoxicity quantification of culture supernatants from E. faecalis -infected macrophages at 2, 6 and 20 hpi using the antibiotic protection assay. Bars represent mean ± SD from n = 5. Statistical significance against WT was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. A subset of this data comparing macrophage cytotoxicity from WT infection at 2, 6 and 20 hpi is shown in . (B-C) Quantification of extracellular egressed bacteria from culture supernatants of RAW264.7 macrophages infected with (B) fsrABDC/gelE deletion mutants and (C) gelE -complemented OG1RF strains at 6 to 12 hpi. Each data point represents mean ± SD of n = 3-4. At each timepoint, statistical significance against WT was assessed using two-way ANOVA with Dunnett’s multiple comparisons test. LOD = limit of detection. (D) Representative Z-projections of WT- and Δ gelE -infected RAW264.7 macrophages at 6, 9 and 12 hpi from (B) , captured with confocal microscopy and stained for Enterococcus specific Group D antigen (yellow), dsDNA (cyan), and F-actin (magenta) (n = 2). Orthogonal Z-axis projections along the blue and red lines are shown in the adjacent colored boxes. White and yellow arrows indicate matched top view and Z-axis projections of selected macrophages with dense intracellular E. faecalis . For all graphs, only comparisons with p < 0.05 are annotated. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Infection, Bacteria, Confocal Microscopy, Staining

Journal: PLOS Pathogens

Article Title: Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

doi: 10.1371/journal.ppat.1013738

Figure Lengend Snippet: (A-B) Total CFU from wound homogenates of (A) mono-infection or (B) competitive infection at 1, 3, and 5 dpi. Median of 3-5 animals per group from one independent experiment is shown. Dotted lines show inoculum CFU for mono-infection. (C) Flow cytometry analysis of intracellular E. faecalis (stained for Group D antigen) in various immune cells at 5 dpi. Median from 6-9 animals per group from 2 independent experiments is shown. (D-E) Quantification of (D) intracellular CFU at 1, 3 and 5 dpi or (E) total CFU at 5 dpi from enzymatically dissociated wounds. Median from 4-5 animals per group from one independent experiment is shown. For A-D, only comparisons with p < 0.05 are annotated. At each timepoint, statistical significance between infection groups was assessed using Mann-Whitney test, except for C, which was assessed using Kruskal-Wallis test with Dunn's multiple comparisons test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001. n.s. = not significant.

Article Snippet: Intracellular E. faecalis was labelled with 1:500 rabbit anti-Streptococcus Group D polyclonal antibody (anti-AgD; American Research Products, Cat# 12-6231D, USA) for 15 min at room temperature, followed by 1:1000 Alexa Fluor 488-conjugated goat anti-Rabbit IgG (Invitrogen, Cat# A11034, USA) for 15 min at room temperature.

Techniques: Infection, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Cell Host & Microbe

Article Title: A combination of cross-neutralizing antibodies synergizes to prevent SARS-CoV-2 and SARS-CoV pseudovirus infection

doi: 10.1016/j.chom.2021.04.005

Figure Lengend Snippet:

Article Snippet: Sf9 (Cat# CRL-1711, RRID: CVCL_0549) and High five cells (Thermo Fisher Scientific Cat# B85502, RRID: CVCL_C190) were cultured in Insect-XPRESS protein-free insect cell medium (Lonza Bioscience Cat# 12-730Q) according to the manufacturer’s instructions and used for generating baculoviruses and for expression of SARSr RBDs for crystallization and binding assays.

Techniques: Expressing, Saline, Recombinant, Transfection, Crystallization Assay, Cloning, Gel Extraction, Electron Microscopy, Software, Staining