Effect of hormones on genes related to hormone receptors and milk component synthesis in bovine mammary epithelial cells grown in two and three dimensional culture system

Figures & data

Figure 1. Morphology of 2D and 3D cultured bovine mammary epithelial cells (BMECs). Note: (A, B) Morphology of two-dimensional cultured bovine mammary epithelial cells with/without PIG (100×). (C, D). Morphology of three-dimensional cultured bovine mammary epithelial cells with/without PIG (100×). PIG: prolactin + insulin + growth hormone.

Figure 2. Immunofluorescence of cytokeratin 18 (100×). Note: Bovine mammary epithelial cells (A); Bovine mammary fibroblasts (B).

Table 1. Primer sequences and PCR parameters.

Genes	GenBank no.	Primer sequences (5′–3′)	Length (bp)	Ta (°C)	Slope	PCR efficiency (%)
EIF3K	NM_001034489	F. CCAGGCCCACCAAGAAGAA	125	60	−3.44	95.28
		R. TTATACCTTCCAGGAGGTCCATGT
MRPL39	NM017446	F. AGGTTCTCTTTTGTTGGCATCC	101	60	−3.24	103.52
		R.TTGGTCAGAGCCCCAGAAGT
UXT	NM_001037471	F.CAGCTGGCCAAATACCTTCAA	125	60	−3.01	114.54
		R.GTGTCTGGGACCACTGTGTCAA
CSN1S1	NM_181029	F.ACATCCTATCAAGCACCAAGGACTC	192	60	−3.06	111.96
		R. GACGAAATGCTTTCAGCTTCCA
CSN2	M-64755.1	F. TCTGCCTCTGCTCCAGTCTT	116	60	−2.925	119.77
		R. AGGAGGGGGCATTCACTTT
CSN3	NM_174294	F. CCAGGAGCAAAACCAAGAAC	148	60	−2.93	119.25
		R. TGCAACTGGTTTCTGTTGGT
MTOR	XM_001788228	F. TGAACTGGAGGCTGATGGACAC	83	60	−3.192	105.71
		R. TGACTGGCCAGCAGAGTAGGAA
EIF4EBP1	BC120290	F. GGCAGGCGGTGAAGAGTC	302	60	−3.410	96..45
		R. CCTGGGCTGCGGGAT
RPS6K1	NM_205816.1	F. ATGAAAGCATGGACCATGGG	101	60	−3.318	100.15
		R. CCGGTATTTGCTCCTGTTAC
STAT5A	NM_001012673	F. AAGACCCAGACCAAGTTCGC	422	60	−3.403	96.73
		R. AGCACCGTGGCAGTAGCAT
AKT1	NM_005163	F: CCT GCT CTC TGG GCT ACT CAA	180	60	−3.340	99.24
		R: CAC GAT GCT GGC GAA GAA
ACC	NM_174224	F：GTTTGTGATGGGCGTGAACC’	101	60	−3.336	99.41
		R：CAGTCTTCTGGGTGGCAGTGAT’
FASN	NM_001012699	F：GTTTGTGATGGGCGTGAACC	85	60	−3.425	95.87
		R：CAGTCTTCTGGGTGGCAGTGAT
DGAT2	NM_205793	F：CATGAAGACCCTCATAGCCG	101	60	−3.342	99.16
		R：ACCAGCCAGGTGAAGTAGAGC’
SCD	AY241993	F：TCCTGTTGTTGTGCTTCATCC	101	60	−3.240	103.52
		R：GGCATAACGGAATAAGGTGGC
FABP3	DN518905	F：GAACTCGACTCCCAGCTTGAA	102	60	−3.321	100.03
		R：AAGCCTACCACAATCATCGAAG
PPARG	NM_181024	F：ATGTCTCATAATGCCATCAGGTT	101	60	−3.400	96.83
		R：GATAACAAACGGTGATTTGTCTGTC
SREBP1	NM_001113302	F: CGC TCT TCC ATC AAT GAC AA	334	60	−3.279	101.84
		R: TTC AGC GAT TTG CTT TTG TG
PLR	NM_174155	F：GATGACTGTGAGGACCAGCA	108	60	−3.362	98.36
		R：AAGGCCATGTGGAAGATTTG
INSR	XM_002688832.2	F：GAGTCTTCACCACCTCTTCA	120	60	−3.345	99.03
		R：TGAGCAGGTCCACGATCT
GHR	AY748827.1	F：CGTCTCTGCTGGTGAAAACA	101	60	−3.33	99.55
		R：AACGGGTGGATCTGGTTGTA
IGF-IR	NM_001244612.1	F： GCTCTGGCCCACGAGTGGAGA	101	60	−3.188	105.90
		R：GCCCTCGATCACCGTGCAGTT

Note. Ta: annealing temperature; UXT: ubiquitously expressed transcript; MRPL39: mitochondrial ribosomal protein L39; EIF3K: eukaryotic translation initiation factor 3 subunit K; FASN: fatty acid synthase; ACC: acetyl-CoA carboxylase; SCD: stearoyl-CoA desaturase; DGAT2: diacylglycerol acyltransferase 2; FABP3: fatty acid-binding protein 3; SREBF1: sterol regulatory element-binding transcription factor; PPARG: peroxisome proliferative activated receptor gamma; CSN1S1: casein alpha s1; CSN2: Casein beta; CSN3: Casein kappa; MTOR: mechanistic target of rapamycin kinase; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; RPS6K1: ribosomal protein S6 kinase; STAT5: Stat5 signal transducer and activator of transcription 5; AKT1: Serine/threonine kinase 1; PRLR: Prolactin receptor; INSR: Insulin receptor; GHR: Growth hormone receptor; IGF-IR: Insulin like growth factor 1 receptor.

Figure 3. Effect of hormones and culture model on triglyceride (TG) content in 2D and 3D cultured bovine mammary epithelial cells (BMECs). Note: Values represent means ± standard error (n = 3 replicates per treatment). ^a,b,c,dMeans with different superscripts differ significantly from each other (p <.05). p Values for culture model, hormones, and model × hormones are based on ANOVA.M: culture model; H: hormone and PIG: prolactin + insulin + growth hormone.

Figure 4. Effect of hormones and culture model on the expression of genes involved in milk fat synthesis in 2D- and 3D-cultured bovine mammary epithelial cell (BMECs). Note: Values represent means ± standard error (n = 3 replicates per treatment). ^a,b,c,dMeans with different superscripts differ significantly (p <.05). P-values for culture model, hormones, and model × hormones are based on ANOVA. M: culture model; H: hormone and PIG: prolactin + insulin + growth hormone; UXT: ACC: acetyl-CoA carboxylase; FASN: fatty acid synthase; SCD: stearoyl-CoA desaturase; DGAT2: diacylglycerol acyltransferase 2; SREBF1: sterol regulatory element-binding transcription factor; PPARG: peroxisome proliferative activated receptor gamma; FABP3: fatty acid-binding protein 3.

Figure 5. Effect of hormones and culture model on the expression of genes involved in milk protein synthesis in 2D- and 3D cultured bovine mammary epithelial cells (BMECs). Note: Values represent means ± standard error (n = 3 replicates per treatment). ^a,b,c,dMeans with different superscripts differ significantly (p < .05). P-values for culture model, hormones, and model × hormones are based on ANOVA. M: culture model; H: hormone and PIG: prolactin + insulin + growth hormone; CSN1S1: casein alpha s1; CSN2: Casein beta; CSN3: Casein kappa; MTOR: mechanistic target of rapamycin kinase; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; RPS6K1: ribosomal protein S6 kinase; STAT5: Stat5 signal transducer and activator of transcription 5; AKT1: Serine/threonine kinase 1.

Figure 6. Effect of hormones and culture model on the expression of genes encoding hormone receptors in 2D- and 3D-cultured bovine mammary epithelial cell (BMECs). Note: Values represent means ± standard error (n = 3 replicates per treatment). ^a,b,c,dMeans with different superscripts differ significantly (p <.05). p Values for culture model, hormones, and model × hormones are based on ANOVA. M: culture model; H: hormone and PIG: prolactin + insulin + growth hormone; PRLR: Prolactin receptor; INSR: Insulin receptor; GHR: Growth hormone receptor; IGF-IR: Insulin like growth factor 1 receptor.