Journal: Regenerative Therapy

Article Title: Orexin-A increases the differentiation of human olfactory sensory neurons through orexin receptor type 1

doi: 10.1016/j.reth.2024.10.014

Figure Lengend Snippet: Immunofluorescence of orexin receptors and dose-response analyses of orexin-A. (a) Immunofluorescence images of OSNs stained with OX1R and OX2R (green). (b) Schematic showing the proposed mechanism of orexin-mediated differentiation of OSNs and dose-response analyses of suvorexant. Western blot densitometric analysis of (c) ASCL1, (d) βIII Tubulin, and (e) OMP, respectively. (p > 0.05, compared to controls, n = 5).

Article Snippet: The orexin receptor type 1 (OX1R) antagonist, SB-674042 (HY-10898, MedChem Express, NJ, USA), and orexin receptor type 2 (OX2R) antagonist, TCS-OX2-29 (HY-100452, MedChem Express) were diluted in the dimethyl sulfoxide, DMSO with 1 μg/mL and 10 μg/mL, respectively.

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Regenerative Therapy

Article Title: Orexin-A increases the differentiation of human olfactory sensory neurons through orexin receptor type 1

doi: 10.1016/j.reth.2024.10.014

Figure Lengend Snippet: The effect of OX1R antagonists (SB674042) and OX2R antagonists (TCS-OX2-29) on the differentiation of OSN. The expression of various markers was assessed through western blots (a and g) and subsequently analyzed densitometrically for ADCY3, Golf, ASCL1, βIII tubulin, and OMP (b-f and h-l). (Asterisks indicate p < 0.05, compared to controls, n = 5–6).

Article Snippet: The orexin receptor type 1 (OX1R) antagonist, SB-674042 (HY-10898, MedChem Express, NJ, USA), and orexin receptor type 2 (OX2R) antagonist, TCS-OX2-29 (HY-100452, MedChem Express) were diluted in the dimethyl sulfoxide, DMSO with 1 μg/mL and 10 μg/mL, respectively.

Techniques: Expressing, Western Blot

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 1. Changes of 11-HSD1, cortisol and LOX in human amnion tissue at parturition and reciprocal regulation of 11- HSD1 and LOX by cortisol in human amnion tissue explants. (A and B) Comparison of 11-HSD1 protein, cortisol (A) and LOX activity (B) between amnion tissues collected at term after elective Cesarean section (term nonlabor) and after vaginal delivery with spontaneous rupture of the membranes (term labor). n 7 for 11-HSD1, n 6 for LOX and n 10 for cortisol. (C) Effects of cortisol (1 M) on 11-HSD1 and LOX protein abundance in cultured human amnion tissue explants. n 3. The top panels of A and C are the representative immunoblots. Data are the mean SEM. Statistical analysis was performed using unpaired (A, B) and paired (C) Student’s t test. *P .05, **P .01 vs. nonlabor or control.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Comparison, Activity Assay, Quantitative Proteomics, Cell Culture, Western Blot, Control

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 2. Distribution of LOX and 11-HSD1 in human fetal membranes and the effects of cortisol in human amnion epithelial and fibroblast cells. (A-C) Immunofluorescence staining of 11-HSD1 (A, orange) and LOX (B, green) in human fetal membranes. Yellow color after merge represents the colocalization of 11-HSD1 and LOX (C). Nuclei were counterstained blue with DAPI. Scale bar, 10 m. (D) Immunofluorescent staining of LOX and LOX activity in cultured amnion epithelial and fibroblast cells. The top panel is the double staining of LOX (green) and vimentin (red) in amnion fibroblasts, and LOX (green) and cytokeratin (red) in amnion epithelial cells. Nuclei were counterstained blue with DAPI. Arrows indicate amnion epithelial cells stained positive for LOX. Scale bar, 10 m. The bottom panel illustrates the effect of cortisol (1 M) on LOX activity in amnion fibroblasts and epithelial cells. n 3. E, The reciprocal effects of cortisol (1 M) on 11-HSD1 and LOX protein abundance in human amnion epithelial and fibroblast cells. (F-H) Immunofluorescent double staining of 11-HSD1 (F, orange) and LOX (G, green) in human amnion fibroblasts. Nuclei were counterstained blue with DAPI. Yellow color after merge represents the colocalization of 11- HSD1 and LOX (H). Scale bar, 10 m. (I and J) Concentration-dependent induction of 11-HSD1 (I, n 5) and reduction of LOX (J, n 5 for mRNA, n 7 for protein) by cortisol (0.01–1 M) in human amnion fibroblasts. The top panels of E, I and J are the representative immunoblots. ae: amnion epithelial cells; af: amnion fibroblasts; ct: chorion trophoblasts. Data are the mean SEM. Statistical analysis was performed with paired Student’s t test (E, K) or one-way ANOVA test (I and J). *P .05, **P .01, ***P .001 vs. control or 0 M.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Immunofluorescence, Staining, Activity Assay, Cell Culture, Double Staining, Quantitative Proteomics, Concentration Assay, Western Blot, Control

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 3. Involvement of 11-HSD1 in the down-regulation of LOX by cortisone in human amnion fibroblasts. (A) Attenuation of cortisone (5 M)-induced reduction in LOX mRNA and protein by 11-HSD1 inhibitor CBX (1 M). n 3. (B) Attenuation of cortisone (5 M)-induced reduction in LOX mRNA and LOX protein by 11-HSD1 inhibitor 10j (1 M). n 3 for mRNA, n 4 for protein. (C) Attenuation of cortisone (5 M)-induced reduction in LOX mRNA, protein and activity by siRNA (50 nM)-mediated knock-down of 11-HSD1. n 5. The top panels of A-C are the representative immunoblots. Data are the mean SEM. Statistical analysis was performed with one-way ANOVA test. *P .05, ***P .001 vs. control or negative scrambled siRNA control; #P .05, ##P .01, ###P .001 vs. cortisone alone.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Activity Assay, Knockdown, Western Blot, Control

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 6. Involvement of GR and nGRE in the inhibition of LOX promoter activity by cortisol and the working model illustrating LOX inhibition in human amnion fibroblasts. (A) Inhibition of LOX promoter (-850bp 126bp, -450bp 126bp) activities by cortisol (1 M). n 4. (B) Blockade of cortisol (1 M)-induced inhibition of LOX promoter (-850bp 126bp) activity by RU486 (1 M, n 4) or siRNA (50 nM, n 5)-mediated knock-down of GR. (C) Enhanced enrichment of GR in LOX promoter spanning the putative nGRE upon cortisol (1 M) stimulation of human amnion fibroblasts as measured with the ChIP assay. n 4. TSS: transcription start site, Fwd and Rev indicate the aligning position of the forward primer (Fwd) and reverse primer (Rev). (D) Abolition of cortisol (1 M)-induced inhibition of LOX promoter (-850bp 126bp) activity by introduction of nucleotide mutations (nucleotides in red) into the putative nGRE (-260 to -252bp) but not by nucleotide mutations in the adjacent CRE (-332 to -325bp) (nucleotides in red). n 3. Data are the mean SEM. Statistical analysis was performed with paired Student’s t test (A, C and D) or one-way ANOVA test (B). *P .05, **P .01 vs. control or negative scrambled siRNA control; #P .05 vs. control or cortisol alone. (E) A working model illustrating the participation of the feed-forward regeneration of cortisol by 11-HSD1 in the structural remodeling in human amnion. Toward the end of gestation, accumulation of cortisol regenerated by 11-HSD1 in the amnion not only induces 11-HSD1 expression, but also down-regulates LOX expression via GR-mediated inhibition of LOX expression in amnion fibroblasts. At the same time, accumulation of cortisol also up-regulates PGE2 production via induction of cPLA2 and COX-2 expression. Increased PGE2 production, in turn, inhibits LOX expression via EP2/EP4 receptors in amnion fibroblasts. Cortisol and PGE2 may act in a coordinated fashion to cause dramatic down-regulation of LOX expression toward the end of gestation resulting in the reduction of collagen cross-linking and weakened tensile strength of the amnion. Reduction in collagen cross-linking may also predispose it to degradation by MMP, which further reduces the tensile strength of the amnion. All of these effects may be implicated in ROM during normal parturition in humans. Preterm activation of this feed-forward mechanism may result in PPROM and preterm birth. Blue arrows indicate stimulation and red lines indicate inhibition. E, cortisone; F, cortisol.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Inhibition, Activity Assay, Knockdown, Control, Expressing, Activation Assay