Journal: Placenta

Article Title: Aldehyde dehydrogenase isoforms and inflammatory cell populations are differentially expressed in term human placentas affected by intrauterine growth restriction

doi: 10.1016/j.placenta.2019.03.015

Figure Lengend Snippet: Western blotting was performed in whole placental samples to quantitate placental ALDH isoform expression in AGA and IUGR pregnancies. ALDH1 and ALDH2 are expressed within the placenta, but no statistical differences were observed between AGA and IUGR pregnancies (ALDH1: AGA 0.71±0.2 vs. IUGR 0.58±0.2; n=5/group; p=0.63 by Student’s t-test) (ALDH2: AGA 0.80±0.2 vs. IUGR 0.54±0.1; p=0.28 by Student’s t-test). ALDH3 expression was not detected by western blot analysis.

Article Snippet: Mouse anti-ALDH1 (cat #611194, BD Biosciences, San Jose, CA, USA) was used at a dilution of 1:200; ALDH2 (cat # ab108306, Abcam, Cambridge, MA, USA) was used at a dilution of 1:100; ALDH3 (cat #ab103895, Abcam) was used at a dilution of 1:100.

Techniques: Western Blot, Expressing

Journal: Placenta

Article Title: Aldehyde dehydrogenase isoforms and inflammatory cell populations are differentially expressed in term human placentas affected by intrauterine growth restriction

doi: 10.1016/j.placenta.2019.03.015

Figure Lengend Snippet: Term human placentas were stained for ALDH1 and ALDH2 expression, and representative sections of Hofbauer cells pictured here. Scale bars represent 100 μm in the top row and 50 μm in the inset panels. Arrows indicate positively stained cells (brown), with magnified insets included in the upper right hand corners for ALDH1 staining. (A) ALDH1 and (B) ALDH2 expression are both detected in placental Hofbauer cell populations, confirmed by CD11c staining in consecutive sections. (A) Positivity of ALDH1 staining by Histologic scoring in Hofbauer cells did not differ in AGA and IUGR groups (AGA: median=2, interquartile range=1–2.5, n=13; IUGR: median=2.25, interquartile range 1.375–2.625, n=10; p=0.38 by Mann-Whitney U testing. (B) Staining of Hofbauer cells with ALDH2 was twice as high in IUGR placentas compared to the AGA group (AGA: median=0, interquartile range-0–2, n=7; IUGR: median=2, interquartile range 0.5–2.375, n=4, p=0.33 by Mann-Whitney U testing). Data are represented in scatter plots with horizontal lines representing median and interquartile range values.

Article Snippet: Mouse anti-ALDH1 (cat #611194, BD Biosciences, San Jose, CA, USA) was used at a dilution of 1:200; ALDH2 (cat # ab108306, Abcam, Cambridge, MA, USA) was used at a dilution of 1:100; ALDH3 (cat #ab103895, Abcam) was used at a dilution of 1:100.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Placenta

Article Title: Aldehyde dehydrogenase isoforms and inflammatory cell populations are differentially expressed in term human placentas affected by intrauterine growth restriction

doi: 10.1016/j.placenta.2019.03.015

Figure Lengend Snippet: Term human placentas were stained for ALDH1 and ALDH2 expression, and representative sections of the decidual stromal cells pictured here. Vimentin staining in serial sections was used to confirm cell populations of interest. Scale bars =100 μm. Arrows indicate positively stained cells (brown). (A) ALDH1 expression trended lower in decidual stromal cells of IUGR-associated placenta compared to AGA placentas (AGA: median=3, interquartile range=3–3, n=12; IUGR: median=2.75, interquartile range: 2.375–3, n=6; p=0.06 by Mann-Whitney U testing). (B) Positivity of ALDH2 staining in decidual stromal cells was not different between groups (AGA: median=3, interquartile range 3–3, n=7; IUGR: median=3, interquartile range 2.625–3, n=4; p=0.36 by Mann-Whitney U testing). Data are represented in scatter plots with horizontal lines representing median and interquartile range values.

Article Snippet: Mouse anti-ALDH1 (cat #611194, BD Biosciences, San Jose, CA, USA) was used at a dilution of 1:200; ALDH2 (cat # ab108306, Abcam, Cambridge, MA, USA) was used at a dilution of 1:100; ALDH3 (cat #ab103895, Abcam) was used at a dilution of 1:100.

Techniques: Staining, Expressing, MANN-WHITNEY

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: Animals : an Open Access Journal from MDPI

Article Title: Expression of Enzymes Associated with Prostaglandin Synthesis in Equine Conceptuses

doi: 10.3390/ani11041180

Figure Lengend Snippet: Primers used for qualitative RT-PCR.

Article Snippet: Polyclonal antibodies directed against COX-1, COX-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX-1 (C20): sc-1752, dilution 1:500; COX-2 (C-20): sc-1745, dilution 1:500, both from goat), previously proven to work in the horse [ ], and against mPGES-1 and cPGES, proven to be specific to equine [ ] (Cayman Chemical Company, Ann Abor, MI, USA), Prostaglandin E Synthase-1 (microsomal) polyclonal antibody # 160140, diltution 1:1:150; Prostaglandin E Synthase (cytosolic) polyclonal antibody # 160150, dilution 1:1000, both rabbit), as well as CBR-1 (Thermo Fisher Scientific, # PA5-53799, polyclonal, rabbit, dilution 1:300) were used as primary antibodies.

Techniques: Sequencing, Amplification, Membrane