Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: AD2900 shows antagonistic activities against S1P1, 2, 3, 4, and 5

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques:

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: (A, B) The percentages of S1P1-positive PBMCs after the treatment with different concentrations of AD2900, FTY720, or SEW2871 and at different time points were examined by FACS analysis. S1P1 expression was tested in PBMCs after a 30-min treatment with AD2900 at different concentrations (A) or after a 30-min or 60-min treatment with 100 nM AD2900, FTY720, or SEW2871 (B) . (C) The percentage of CCR7-positive PBMCs was tested by FACS analysis after a 30-min treatment with 100 nM AD2900, FTY720, or SEW2871. All the significances are compared to untreated PBMCs. Results summarize the results of at least four independent experiments. Results of Student’s t -test: *(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques: Expressing, Two Tailed Test

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: C57BL/6 mice were orally administered with 1.8, 2.7, and 3.6 mg/l AD2900 or 1.8 mg/l FTY720 for 2 days, as shown in Figure . Leukocytes from blood, spleen, and pLNs were collected and stained with CD3e and S1P1 or CCR7 fluorescent antibodies and then analyzed by FACS analysis. The percentages of S1P1+ CD3e+ T cells from blood (A) , spleen (B) , and pLNs (C) are shown. The percentages of CCR7+ CD3e+ T cells from blood (D) , spleen (E) , and pLNs (F) are shown. All the significances are compared to untreated healthy mice. Results summarize at least three independent experiments. Results of Student’s t -test:*(P < 0.05, two-tailed test), ** (P < 0.01, two-tailed test), *** (P < 0.001, two-tailed test), **** (P < 0.0001, two-tailed test).

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques: Staining, Two Tailed Test

Journal: Oncotarget

Article Title: The novel sphingosine-1-phosphate receptors antagonist AD2900 affects lymphocyte activation and inhibits T-cell entry into the lymph nodes

doi: 10.18632/oncotarget.18626

Figure Lengend Snippet: As an antagonist to S1P receptors 1–5, AD2900 can compete with S1P to bind S1P receptors leading to reduced S1P signaling and enhanced expression of S1P1 on T cells in S1P-rich environments such as the blood and the spleen. This altered expression, together with decreased CCR7 expression, inhibits T-cell entry into the lymph nodes (LNs) from the blood, causing accumulation of T cells in the blood. However, the entry of T cells to the spleen is not affected because it is not S1P dependent. Since Tcm-like cells express CCR7, these cells are attracted to the spleen and accumulate in it; yet, S1P1 elevated expression may have an effect on the S1P-dependent ingression of these cells from the MZ to the white pulp. Tef/em-like cells, which are CCR7 negative, are the primary T-cell subpopulation in the blood after AD2900 treatment. The significant decrease in naive T-cell counts in the circulation and peripheral lymphoid tissues tested may be explained by the inhibition of S1P signaling in the thymus leading to attenuated T-cell egression from the thymus to the circulation. Arrow key: thick = response; dashed = inhibition.

Article Snippet: To analyze S1P1 surface expression on AD2900-treated PBMCs, the following were used: anti-S1P1 (Alomone labs, Israel), anti-EDG-1 (Abcam, UK), Dylight405-conjugated AffiniPure Goat Anti-Rabbit lgG (H + L), and APC-conjugated AffiniPure F(ab')2 Goat Anti-Rabbit lgG(H + L) (Jackson ImmonoResearch, USA).

Techniques: Expressing, Inhibition

Journal: Stem cell research

Article Title: Generation of induced pluripotent stem cells-derived hepatocyte-like cells for ex vivo gene therapy of primary hyperoxaluria type 1.

doi: 10.1016/j.scr.2019.101467

Figure Lengend Snippet: Fig. 5. Characterization of hepatocyte-like cells (HLCs) and analysis of AGT expression rescue. (A) Bright field and immunofluorescence staining of endodermic (SOX17) and hepatic markers (HNF4a and aFP) throughout hepato- cyte differentiation. Nuclei were localized by DAPI (blue). Scale bars, 100 μm. Magnification, 100X. (B) RT-qPCR analysis of hepatic gene markers in HLCs at day 31 of hepatocyte differentiation. Gene expression was normalized to the endogenous HuP0 gene and data are means ± SD of four independent experiments. (C) CYP3A4 activity analysis in HLCs at day 31 of differentiation. Data were expressed as means ± SD of three independent experiments;,٭p < 0.05. (D) RT-PCR analysis of eGFP and endogenous (endo. AGXT) or exogenous AGXT expression in HLCs. (E) Western blotting analysis of AGT protein ex- pression in HLCs. ND; not determined. Abbreviations: aFP, alpha-fetoprotein; a1AT, alpha-1 antitrypsin; ns, non-significant; endo., endogenous. (For interpretation of the refer- ences to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: At day 7, the DECs were enzymatically dissociated with TrypLETM Select Enzyme (Life Technologies), reseeded into appropriate cell culture vessels, and differentiated into HLCs using the Cellartis® Hepatocyte Differentiation Kit (Takara Bio Europe), according to the manufacturer's instructions.

Techniques: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Gene Expression, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot