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01.12.2019 | Research | Ausgabe 1/2019 Open Access

Journal of Ovarian Research 1/2019

Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro

Zeitschrift:
Journal of Ovarian Research > Ausgabe 1/2019
Autoren:
Huali Chen, Youfu Yang, Youlin Wang, Yuan Li, Yamei He, Jiaxin Duan, Dejun Xu, Yifei Pei, Jianyong Cheng, Li Yang, Rongmao Hua, Xiaoya Li, Jie Wang, Xiaohan Jiang, Huanshan He, Lin Wu, Dingbang Liu, Qingwang Li
Wichtige Hinweise

Supplementary information

Supplementary information accompanies this paper at https://​doi.​org/​10.​1186/​s13048-019-0567-4.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Abstract

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 μM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCβ1, protein kinase C β (PKCβ), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calciumsensitive targets and several downstream genes might take part in the processes.
Zusatzmaterial
Additional file 1: Figure S1. Effect of U73122 on the mRNA abundance of PLCB1 in porcine granulosa cells. Cells were challenged with the doses of U73122(from 0 μM to 5 μM) for the times given. Data are mean ± S.E.M.of three independent replicates. For each treatment, means without common letters are significantly different(p<0.05). Figure S2. Effect of m3M3FBS on the mRNA abundance of PLC in porcine granulosa cells. Cells were challenged with the doses of m3M3FBS (from 0 μM to 50 μM) for the times given. Data are mean ± S.E.M.of three independent replicates. For each treatment, means without common letters are significantly different(p<0.05).Figure S3. One representative scatter diagram at each time point for apoptosis induced by U73122 measured with annexin V/PI staining in porcine granulosa cells. Cells were challenged with 0.5 μM U73122 for 4 h (gene) or for the times given(percentage of apoptotic cells), which were processed for annexin V/PI staining and measured by flow cytometry assay. Figure S4. One representative scatter diagram at each time point for apoptosis induced by m3M3FBS measured with annexin V/PI staining in porcine granulosa cells. Cells were challenged with 0.5 μM m3M3FBS for 4 h(gene) or for the times given(percentage of apoptotic cells), which were processed for annexin V/PI staining and measured by flow cytometry assay. Figure S5. Fluorescence intensity of intracellular Ca2+ in porcine granulosa cells. The area in the histogram represents the fluorescence intensity variation of granulosa cells measured by Flow cytometry and analyzed by FlowJo V10. A and B indicate the fluorescence intensity treated with DMF (Control) and U73122; C and D indicate the fluorescence intensity cultured with DMSO (Control) and m-3M3FBS.
Literatur
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