Neuron-derived neurotrophic factor promotes the differentiation of intramuscular and subcutaneous adipocytes in goat

Figures & data

Table 1. The tools for analysis and the content of their analysis are listed in the table.

Software or online tools	Analytical content
Primer Primiers 5.0	Primer design
ORF Finder	Open reading frame prediction and amino acid sequence translation
ExPASy ProtParam	Analysis of physicochemical properties of protein
ExPASy	Prediction of protein secondary structure
SWISS-MODEL	Prediction of protein tertiary structure
SignalP4.1 Server	Signal peptide analysis
TMHMM	Prediction of transmembrane domain
PSORTII	Subcellular localization
STRING	Protein interaction analysis
MEGA5.0	Construction of phylogenetic tree
Blast	Homology comparison analysis

Table 2. Information of primers.

Target Gene	Primer sequence	Application	Accession	Length(bp)
NENF	S:GAGCCCATCTACATGGCAGTC A:GTAATTCCACTGCCCACGAA	Cloning	XM_018060749.1
NENF	S: GCAGACCTCACCCATGACA A: GAGGATTCTTCGGGCTGTAT	qPCR	XM_018060749.1	123
AP2	S: GTCCTTCAAATTGGGCCAGGA A: CTGGTGGTAGTGACACCGTT	qPCR	NM_001285623.1	192
CEBPα	S:CCGTGGACAAGAACAGCAAC A:AGGCGGTCATTGTCACTGGT	qPCR	XM_018062278.1	142
CEBPβ	S: CAAGAAGACGGTGGACAAGC A: AACAAGTTCCGCAGGGTG	qPCR	XM_018058020.1	204
SREBP	S: AAGTGGTGGGCCTCTCTGA A: GCAGGGGTTTCTCGGACT	qPCR	NM_001285755.1	127
PREF-1	S: CCTGAAAATGGATTCTGCGACG A: GACACAGGAGCACTCGTACTG	qPCR	NM_001314212.1	255
LPL	S: TCCTGGAGTGACGGAATCTGT A: GACAGCCAGTCCACCACGAT	qPCR	NM_001285607.1	174
PPARγ	S: AAGCGTCAGGGTTCCACTATG A: GAACCTGATGGCGTTATGAGAC	qPCR	NM_001285658.1	197
ADRP	S: TACGATGATACAGATGAATCCCAC A: CAGCATTGCGAAGCACAGAGT	qPCR	>NM_001285596.1	202
ATGL	S: CAAGGAGACGACGTGGAACA A: CATAGATGTGCGTGGCGTTG	qPCR	XM_018042656.1	125
DGAT1	S: CCACTGGGACCTGAGGTGTC A: GCATCACCACACACCAATTCA	qPCR	XM_018058728.1	101
GPAM	S: CCAGTATCCCGTCTTTGGGT A: TTACGTTGGTGGCAAACATGC	qPCR	XM_013975269.2	147
ACC	GGAGACAAACAGGGACCATT ATCAGGGACTGCCGAAAC	qPCR	XM_018064174.1	146
UXT	S: GCAAGTGGATTTGGGCTGTAAC A: ATGGAGTCCTTGGTGAGGTTGT	qPCR	XM_005700842.2	180

Figure 1. Cloning and sequence analysis of NENF. (a) Amplification of NENF gene in Jianzhou Big-eared goat; DNA marker DL2000, NENF target skip. (b) The sequences of nucleotide and deduced amino acid of NENF cDNA in Jianzhou Big-eared goat. The two green arrows indicated the positions of primer pair. The start codon ATG and stop codon TGA are represented by red bases, respectively. (c) The composition of deduced amino acid for NENF protein in Jianzhou Big-eared goat. (d) Nucleotide sequence identity of NENF cDNA between Jianzhou Big-eared goats and other mammalian species.

Figure 2. Structural analysis of NENF protein in goat. (a) Secondary structure prediction of NENF protein, α-helix are represented in blue, extended strand was represented in red, β-turn are represented in green, random coils are represented in orange. (b) Prediction of tertiary structure of NENF protein. (c) NENF protein interaction network.

Figure 3. Relative expression level of NENF in goat tissues and subcutanesous adipocytes and intramuscular adipocytes. (a) Relative expression of NENF during differentiation of intramuscular adipocytes. (b) Relative expression of NENF during differentiation of subcutaneous adipocytes. *significant difference. (c) Relative expression level of NENF in different tissues in goat. ‘A’ means extremely significant difference, ‘B’ means significant difference.

Figure 4. NENF overexpression promotes intramuscular adipocytes differentiation. (a) Quantitative polymerase chain reaction (qPCR) detects the overexpression efficiency of NENF in goat intramuscular adipocytes. (b) Oil red O staining of goat adipocytes. (c) Quantitative analysis of oil red O staining signal was indicated by absorbance at 492 nm. (d) Effect of overexpression of NENF on gene related to adipocytes differentiation of goat, expression changes in AP2, CEBPα, CEBPβ, SREBP-1, PREF-1, and PPARγ in intramuscular adipocytes. (e) Effects of NENF overexpression on genes associated with lipid synthesis in goat adipocytes. Changes in the expression of ADRP, ATGL, DGAT1, GPAM, ACC, and LPL in intramuscular adipocytes. *means P < 0.05, significant difference. **means P < 0.01, extremely significant difference.

Figure 5. NENF overexpression promotes subcutaneous adipocytes differentiation. (a) qPCR detects the overexpression efficiency of NENF in goat subcutaneous adipocytes. (b) Oil red O staining of goat adipocytes. (c) Quantitative analysis of oil red O staining signal was indicated by absorbance at 492 nm. (d) Effect of overexpression of NENF on gene related to adipocytes differentiation of goat, expression changes in AP2, CEBPα, CEBPβ, SREBP-1, PREF-1, and PPARγ in subcutaneous adipocytes. (e) Effects of NENF overexpression on genes associated with lipid synthesis in goat adipocytes. Changes in the expression of ADRP, ATGL, DGAT1, GPAM, ACC, and LPL in subcutaneous adipocytes. *means P < 0.05, significant difference. **means P < 0.01, extremely significant difference.