Journal: The Journal of Reproduction and Development

Article Title: FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation

doi: 10.1262/jrd.2023-015

Figure Lengend Snippet: Expression levels of FABP4 in sheep endometrium. (a) FABP4 mRNA expression in sheep endometrium on day 4 and day 15. (b, c) FABP4 protein expression in sheep endometrium on day 4 and 15. The optical density was normalized to the density of β-actin in the same lane. (d, e) Rabbit IgG group was used as the negative control for analyzing the localization and expression of FABP4 in sheep endometrium. Data are presented as mean ± standard error with significant differences at P < 0.05 and extremely significant differences at P < 0.01. * indicates P < 0.05, ** indicates P < 0.01, and no sign indicates that the difference is not significant. The technique was repeated thrice.

Article Snippet: SEECs were fixed with 4% paraformaldehyde at room temperature, blocked with BSA for 30 min, and FABP4 rabbit polyclonal antibody (Boster, BM4029, 1:50) and CK18 mouse monoclonal antibody (Abcam, Cambridge, UK, ab668, 1:50) were added overnight at 4°C.

Techniques: Expressing, Negative Control

Journal: The Journal of Reproduction and Development

Article Title: FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation

doi: 10.1262/jrd.2023-015

Figure Lengend Snippet: Expression of FABP4 in SEECs. (a) Isolation and purification of SEECs (250 ×). When the cells reached the sixth passage, they became larger and rounder and began to senesce. (b) Immunofluorescence identification of SEECs (CK18) (100 ×). (c) Localization of FABP4 in SEECs (Yellow arrows are FABP4 in the nuclei) (25 ×).

Article Snippet: SEECs were fixed with 4% paraformaldehyde at room temperature, blocked with BSA for 30 min, and FABP4 rabbit polyclonal antibody (Boster, BM4029, 1:50) and CK18 mouse monoclonal antibody (Abcam, Cambridge, UK, ab668, 1:50) were added overnight at 4°C.

Techniques: Expressing, Isolation, Purification, Immunofluorescence

Journal: The Journal of Reproduction and Development

Article Title: FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation

doi: 10.1262/jrd.2023-015

Figure Lengend Snippet: Hormone treatment of SEECs and detection of changes in FABP4 expression. (a) Protein expression levels of PGR and ER after combined hormone treatment. (b) The mRNA expression levels of ISG15, HOXA10, CXCL10, and RSAD2 after hormone treatment. (c) Levels of the prostaglandin secreted from the SEECs after hormone treatment.(d) Expression levels of FABP4 after hormone treatment. All data are presented as mean ± standard error. Differences were considered significant at P < 0.05 and extremely significant at P < 0.01.

Article Snippet: SEECs were fixed with 4% paraformaldehyde at room temperature, blocked with BSA for 30 min, and FABP4 rabbit polyclonal antibody (Boster, BM4029, 1:50) and CK18 mouse monoclonal antibody (Abcam, Cambridge, UK, ab668, 1:50) were added overnight at 4°C.

Techniques: Expressing

Journal: The Journal of Reproduction and Development

Article Title: FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation

doi: 10.1262/jrd.2023-015

Figure Lengend Snippet: FABP4 inhibition impedes SEEC function. (a, b) Mobility of SEECs at 0, 24, 48, 72 and 96 h was measured using a scratch test. Migratory capacity was calculated as a percentage of healing area relative to time 0. (c) CCK-8 viable cell counts quantified the proliferative capacity of SEECs treated with hormone and FABP4 inhibitor BMS309403. (d–g) Expression levels of EMT after hormone and inhibitor treatment (E-cadherin, N-cadherin, Vim, and β-catenin). (h, i) Changes in endoplasmic reticulum stress-related protein CHOP and GRP78 were measured. (j, k) Changes in autophagy-related proteins p-mTOR, LC3B II/I, and P62. Data are expressed as mean ± standard error. Differences were considered significant at P < 0.05 and extremely significant at P < 0.01.

Article Snippet: SEECs were fixed with 4% paraformaldehyde at room temperature, blocked with BSA for 30 min, and FABP4 rabbit polyclonal antibody (Boster, BM4029, 1:50) and CK18 mouse monoclonal antibody (Abcam, Cambridge, UK, ab668, 1:50) were added overnight at 4°C.

Techniques: Inhibition, CCK-8 Assay, Expressing

Journal: The Journal of Reproduction and Development

Article Title: FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation

doi: 10.1262/jrd.2023-015

Figure Lengend Snippet: TG and 3-MA treatment partially restores SEEC function after BMS30940 suppression of FABP4. (a, b) Expression levels of key proteins in the endoplasmic reticulum stress signaling pathway after combined treatment with hormones, BMS309403, and TG. (c, d) Expression levels of key proteins in autophagy and apoptosis signaling pathways after combined treatment with hormones, BMS309403, and 3-MA. (e) Secreted prostaglandin levels from SEECs after combined treatment with hormones, BMS309403, and TG or 3-MA. Data are expressed as mean ± standard error. Differences were considered significant at P < 0.05 and extremely significant at P < 0.01.

Article Snippet: SEECs were fixed with 4% paraformaldehyde at room temperature, blocked with BSA for 30 min, and FABP4 rabbit polyclonal antibody (Boster, BM4029, 1:50) and CK18 mouse monoclonal antibody (Abcam, Cambridge, UK, ab668, 1:50) were added overnight at 4°C.

Techniques: Expressing, Protein-Protein interactions

Journal: Frontiers in Pharmacology

Article Title: Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes

doi: 10.3389/fphar.2019.01695

Figure Lengend Snippet: A greater proportion of MACS-derived adipose-derived stem cell (ASC) exhibit activity in in vitro differentiation assays compared to culture-derived ASC. (A) MACS-derived and culture-derived ASC were subjected to an in vitro adipogenic differentiation assay. The graph represents the percentage of cells which stained positive for FABP4 expression after quantification. Panel (C) shows representative images from the adipogenic differentiation assay for three donors (D1–D3) where FABP4 positive cells are stained green and nuclei are stain blue. The top row contains images of MACS-derived ASC and the bottom row contains images from culture-derived ASC. The graph in (B) represents the percentage of cells which stained positive for alizarin red after 3 weeks of culture in commercial osteogeneic differentiation media. Panel (D) shows representative images from the alizarin red assay for three donors (D1–D3). The top row contains images of MACS-derived ASC and the bottom row contains images from culture-derived ASC. * denotes a p value of < 0.05.

Article Snippet: Cells were then subjected to immunocytochemistry using a 1:200 dilution of rabbit antihuman FABP4 polyclonal antibody (Cat #10004944, Cayman Chemicals) and then incubated with a 1:200 dilution Alexa Fluor ® 488 conjugated goat antirabbit IgG secondary antibody (Cat # A11008, Molecular Probes ® ) and 1:2,000 diluted DAPI (Cat# D3571, Molecular Probes ® ).

Techniques: Derivative Assay, Activity Assay, In Vitro, Differentiation Assay, Staining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes

doi: 10.3389/fphar.2019.01695

Figure Lengend Snippet: Ex vivo MACS-derived adipose-derived stem cell (ASC) exhibit rapid and marked changes in gene expression when subjected to standard tissue culture conditions. MACS-derived ASC were subjected to standard tissue-culture conditions and microarray analysis was performed at day 0, day 3, or day 28 post sort. (A) A heatmap was generated using a list of all transcripts which exhibited a fold change >10 when microarray data was analyzed using the Affymetrix Transcript Analysis Console software. Blue represents downregulated genes and red upregulated genes from three donor samples (1–3) at day 0, day 3, or day 28 post sort. (B) Unsupervised clustering was performed using all transcripts detected in the microarray data when processed using robust microarray average (RMA). This was then plotted as a dendogram to pictorially represent the relationship between the three donor samples (1–3) at day 0, day 3, or day 28 post sort. (C) Real-time PCR was used to validate a subset of the microarray data (target genes SCRG1, POSTN, KLF4, GREM1, C-MYC, OGN, MGP, CXCL14, FABP4, SOX2, and OCT4 as indicated) in at least four, and a maximum of eight subsequent donors (D1–D8). * denotes a p value of < 0.05, *** denotes a p value of < 0.001.

Article Snippet: Cells were then subjected to immunocytochemistry using a 1:200 dilution of rabbit antihuman FABP4 polyclonal antibody (Cat #10004944, Cayman Chemicals) and then incubated with a 1:200 dilution Alexa Fluor ® 488 conjugated goat antirabbit IgG secondary antibody (Cat # A11008, Molecular Probes ® ) and 1:2,000 diluted DAPI (Cat# D3571, Molecular Probes ® ).

Techniques: Ex Vivo, Derivative Assay, Gene Expression, Microarray, Generated, Software, Real-time Polymerase Chain Reaction