Acta Veterinaria et Zootechnica Sinica ›› 2024, Vol. 55 ›› Issue (10): 4391-4402.doi: 10.11843/j.issn.0366-6964.2024.10.013
• Animal Genetics and Breeding • Previous Articles Next Articles
Lan FENG1(
), Xue FENG1, Yulin MA1, Lingkai ZHANG1, Yanfen MA1, Dawei WEI1, Fen LI2, Lupei ZHANG3, Runjun YANG4, Yun MA1,*(), Bei CAI1,*()
Received:2024-03-25
Online:2024-10-23
Published:2024-11-04
Contact:
Yun MA, Bei CAI
E-mail:15202683541@163.com;mayun@nxu.edu.cn;caibei1115@163.com
CLC Number:
Lan FENG, Xue FENG, Yulin MA, Lingkai ZHANG, Yanfen MA, Dawei WEI, Fen LI, Lupei ZHANG, Runjun YANG, Yun MA, Bei CAI. Study on the Function of PPP5C Gene in Regulating the Proliferation and Differentiation of Bovine Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4391-4402.
Table 1
siRNA sequence information"
| 名称 Name | 上游引物序列(5′→3′) Upstream primer sequence | 下游引物序列(5′→3′) Downstream primer sequence |
| Si-PPP5C-425 | GGUGAAGGUGAAGCCACAUTT | AUGUGGCUUCACCUUCACCTT |
| Si-PPP5C-610 | GCAGGGUGACAAUCACCUUTT | AAGGUGAUUGUCACCCUGCTT |
| Si-PPP5C-1081 | CCCAGUGCAUCAAUGGCAATT | UUGCCAUUGAUGCACUGGGTT |
| 对照Control | UUCUCCGAACGUGUCACGUTT | ACGUGACACGUUCGGAGAATT |
Table 2
Marker genes and internal reference gene amplification primers sequence information"
| 基因 Gene | 上游引物序列(5′→3′) Upstream primer sequence | 下游引物序列(5′→3′) Downstream primer sequence |
| CDK1 | GAAGGGGTTCCTAGTACTGC | ATGAACTGACCAGGAGGG |
| CDK2 | ATGAACTGACCAGGAGGG | GCCAGGAGTTACTTCTATGC |
| PCNA | GAACCTCACCAGCATGTCCA | TACTAGTGCCAACGTGTCCG |
| PPARγ | AGGATGGGGTCCTCATATCC | GTCAGCTCTTGGGAACGGAA |
| C/EBPα | TGGACAAGAACAGCAACGAG | TTGTCACTGGTCAGCTCCAG |
| FABP4 | AAGTCAAGAGCATCGTAA | CCAGCACCATCTTATCAT |
| GAPDH | CCAACGTGTCTGTTGTGGAT | CTGCTTCACCACCTTCTTGA |
Fig. 1
Adipocytes isolation and culture A. Adipocytes separation, 7 d; B. Cell passaging culture, 1 d; C. Cell passaging culture, 3 d"
Fig. 2
Constructing a model of bovine precursor adipocyte differentiation and PPP5C temporal expression A. Oil red O staining of bovine precursor adipocytes at 0 and 6 d of differentiation; B. BODIPY staining of bovine precursor adipocytes at 0 and 6 d of differentiation; C. Detection of mRNA expression levels of C/EBPɑ in bovine precursor adipocytes differentiated at 0, 2, 4, 6 and 8 d by qRT-PCR; D. qRT-PCR was performed to detect the mRNA expression level of PPARγ in bovine precursor adipocytes at 0, 2, 4, 6 and 8 d of differentiation; E. qRT-PCR was performed to detect the mRNA expression level of PPP5C in bovine precursor adipocytes at 0, 2, 4, 6 and 8 d of differentiation. The data were expressed as "mean ±SEM", n=3, *P < 0.05, **P < 0.01, the same as below"
Fig. 3
PPP5C gene overexpression, interference efficiency assay A. The mRNA expression levels of PPP5C in bovine precursor adipocytes were detected by qRT-PCR after transfection with pcDNA3.1 (Control) and pcDNA3.1-PPP5C, respectively; B. qRT-PCR was used to detect the mRNA expression level of PPP5C in bovine precursor adipocytes after transfection with si-NC, si-PPP5C-425, si-PPP5C-610 and si-PPP5C-1081, respectively"
Fig. 4
Effect of interfering with PPP5C gene on bovine adipocyte proliferation A. The mRNA expression levels of CDK1, CDK2 and PCNA in bovine precursor adipocytes were detected by qRT-PCR after transfection with si-NC, si-PPP5C-425, respectively; B. Flow cytometry to detect the cell cycle of bovine precursor adipocytes after transfection with si-NC, si-PPP5C-425, respectively; C. Flow cytometry was used to detect the cell cycle value added rate of bovine precursor adipocytes after transfection with si-NC and si-PPP5C-425, respectively; D. EdU assay for cell proliferation after transfection of bovine precursor adipocytes with si-NC, si-PPP5C-425, respectively; E. EdU assay for cell proliferation rate of bovine precursor adipocytes after transfection with si-NC, si-PPP5C-425, respectively; F. CCK-8 assay for cell viability of bovine precursor adipocytes after transfection with si-NC, si-PPP5C-425, respectively"
Fig. 5
Effect of overexpression of PPP5C gene on bovine adipocyte proliferation A. The mRNA expression levels of CDK1, CDK2 and PCNA were detected by qRT-PCR in bovine precursor adipocytes transfected with pcDNA3.1 and pcDNA3.1-PPP5C, respectively; B. Flow cytometry was used to detect the cell cycle of bovine precursor adipocytes after transfection with pcDNA3.1, pcDNA3.1-PPP5C, respectively; C. Flow cytometry was used to detect the cell cycle value-added rate of bovine precursor adipocytes after transfection with pcDNA3.1 and pcDNA3.1-PPP5C, respectively; D. EdU assay for cell proliferation after transfection of bovine precursor adipocytes with pcDNA3.1, pcDNA3.1-PPP5C, respectively; E. EdU assay for cell proliferation rate of bovine precursor adipocytes after transfection with pcDNA3.1, pcDNA3.1-PPP5C, respectively; F. CCK-8 assay for cell viability of bovine precursor adipocytes after transfection with pcDNA3.1, pcDNA3.1-PPP5C, respectively"
Fig. 6
Effect of interfering with PPP5C gene on bovine adipocyte differentiation A. The mRNA expression levels of PPARγ, C/EBPα and FABP4 in bovine precursor adipocytes were detected by qRT-PCR after transfection with si-NC, si-PPP5C-425 and differentiation for 4 d, respectively; B. BODIPY staining after transfection of si-NC, si-PPP5C-425 to bovine precursor adipocyte and differentiation for 4 d, respectively; C. Oil red O staining after transfection of si-NC, si-PPP5C-425 to bovine precursor adipocyte and differentiation for 4 d, respectively; D. Nile red staining after transfection of si-NC, si-PPP5C-425 into bovine precursor adipocytes and differentiation for 4 d, respectively; E. TG content was assayed after transfection of si-NC and si-PPP5C-425 into bovine precursor adipocytes and differentiation for 4 d, respectively"
Fig. 7
Effect of overexpression of PPP5C gene on bovine adipocyte differentiation A. The mRNA expression levels of PPARγ, C/EBPα and FABP4 were detected by qRT-PCR in bovine precursor adipocytes transfected with pcDNA3.1, and pcDNA3.1-PPP5C after 4 d of differentiation, respectively; B. BODIPY staining after transfection of pcDNA3.1, pcDNA3.1-PPP5C to bovine precursor adipocyte and differentiation for 4 d, respectively; C. Oil red O staining after transfection of pcDNA3.1, pcDNA3.1-PPP5C to bovine precursor adipocyte and differentiation for 4 d, respectively; D. Nile red staining after transfection of pcDNA3.1, pcDNA3.1-PPP5C to bovine precursor adipocyte and differentiation for 4 d, respectively; E. TG content was assayed after transfection of pcDNA3.1 and pcDNA3.1-PPP5C into bovine precursor adipocytes for differentiation 4 d, respectively"
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