Supplementary MaterialsAdditional file 1: Figure S1. allowed to measure both estradiol and progesterone in supernatants of the same 96-very well JAK-IN-1 dishes. (TIF 342 kb) 12958_2018_357_MOESM3_ESM.tif (342K) GUID:?4F033234-4557-4F5E-AC0E-43AAA82194E2 Extra file 4: Shape S3. Gene manifestation of (A) free of charge fatty acidity receptor 1 (FFAR4 (Accession quantity: NP_001315586.1) no identification with additional amino acidity sequences of bovine proteome. Rabbit IgG was utilized because the control using the same supplementary antibody for FFAR4 recognition. Nuclei had been stained with Hoechst 33,258 (blue fluorescence). Fluorescence Rabbit Polyclonal to COX19 was noticed under a Zeiss confocal microscope LSM700 (Carl Zeiss Microscopy GmbH, Munich, Germany) using an essential oil 63 objective and the correct filters. The pictures had been captured using Zen 2012 software program (black edition edition 8.0, Carl Zeiss Microscopy GmbH). The picture framed in reddish colored is really a magnification of the area framed in red from the original image. Bars?=?10?m. (TIF 132 kb) 12958_2018_357_MOESM5_ESM.tif (132K) GUID:?DC308CE0-B4EB-4AB7-912D-599885F32C85 Additional file 6: Figure S6. Protein expression of (A) proliferating cell nuclear antigen (PCNA), (B) hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), (C) steroidogenic acute regulatory protein (StAR) and (D) JAK-IN-1 cytochrome P450 family 11 subfamily JAK-IN-1 A member 1 (CYP11A1) after 15?h treatment with JAK-IN-1 DHA. Effects of DHA treatment on protein levels were assessed in bovine granulosa cells after 15?h culture in enriched McCoys 5A media in presence or absence of DHA 10 or 20?M. The chemical DMSO alone (1/2000) was used as a negative control due to its solvent activity on DHA. Protein extracts were separated by electrophoresis on 4C12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-PCNA (A), anti-HSD3B1 (B), anti-StAR (C) or anti-CYP11A1 (D) antibodies. The blots were stripped and re-probed with antibodies against Vinculin (VCL). The blots presented are representative of the quantification reported in Fig. ?Fig.6.6. (TIF 180 kb) 12958_2018_357_MOESM6_ESM.tif (180K) GUID:?006FB7E2-61F6-4FCC-8C38-50ADA65CF3A8 Additional file 7: Figure S5. Signaling pathways in bovine granulosa cells after treatment with other concentrations of DHA (50?M) or TUG-891 (10 and 50?M). Effects of DHA or TUG-891 on JAK-IN-1 phosphorylation of (A) mitogen-activated protein kinase 14 (MAPK14), (B) AMP-activated protein kinase (AMPK) and (C) mitogen-activated protein kinase 1/3 (MAPK1/3) signaling pathways were assessed in bovine granulosa cells cultured for 15?h in enriched McCoys 5A media with 50?M DHA or with 10 or 50?M TUG-891, as described in Material and Method section for 5, 10, 30 and 60?min. Protein extracts were separated by electrophoresis on 4C12% (w:v) SDS-polyacrylamide gel. After electrotransfer to nitrocellulose membranes, the proteins were probed with anti-phosphorylated (p-)MAPK14 (A), anti-p-AMPK (B) or anti-p-MAPK1/3 (C) antibodies. The blots were stripped and re-probed with antibodies against MAPK14, AMPK, or MAPK1/3, respectively. Bands on the blots were quantified. Results of four independent experiments are presented as the ratio of p-protein to total protein, normalized from the ratio seen in control at each correct time period and indicated as suggest??SEM, as time passes 0?min getting add up to 1 (for research). Pubs with different superscripts are considerably different (gene abbreviation, item size in foundation pair, primer effectiveness Gene expression evaluation in GCs GCs had been cultured in 48-well meals (2.5??105 viable cells/250?L media/very well) in revised McCoys 5A moderate within the presence or lack of DHA (1, 10, 20 or 50?M) or TUG-891 (1, 10 or 50?M) for 8?h. After removal of the moderate, cells had been retrieved using 200?l/well of TriZol reagent (Invitrogen, Cergy Pontoise, France), frozen and stored until evaluation immediately. Yet another condition, consisting in GC gathered from ovaries and immediately freezing and kept until evaluation was also constituted and called in vivo GC. Total RNA was extracted from.