Fig 2: The effect of PRLR-STAT5 on transcriptional activity of SNAT1/2 regulated by PRL. PRLR: prolactin receptor; STAT5: signal transducers and activators of transcription 5. (A) SNAT1/2 promoter fluorescence activity is measured after PRLR is knocked down. (B) Promoter fluorescence activity of SNAT1/2 in GMECs after knocked down STAT5. (C) Protein and mRNA levels of STAT5 and SNAT1/2 are measured after knockdown of PRLR. (D) Protein and mRNA levels of SNAT1/2 in GMECs after knocked down STAT5. pSNAT1/2-NC: negative control for PRLR/STAT5 siRNAs during SNAT1/2 promoter assay; pSNAT1/2 + siPRLR/siSTAT5: The SNAT1/2 promoter–reporter assay was accompanied by PRLR or STAT5 RNA interference; pSNAT1/2: The SNAT1/2 promoter–reporter assay accompanied with pGL3-basic vector; NC: the negative control for PRLR or STAT5 RNA interference; siPRLR/siSTAT5: siRNAs for PRLR or STAT5. Data are represented as means ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001. Original images can be found in Supplementary Files.

Fig 3: SNAT1/2 nucleic acid level detection. (A) RNA levels of SNAT1/2 are detected by transcriptome sequencing after treatment with 200 ng/mL of PRL. (B) qPCR assays of SNAT1/2 mRNA levels in GMECs after being treated with PRL (0, 100, 200, 400, 800 ng/mL). (C) qPCR assays for mRNA levels of SNAT1/2 in GMECs after being treated with induced lactation system (EGF + ITS + HC) supplemented with PRL (0, 2500, 5000, 7500, 10,000 ng/mL). EGF: epidermal growth factor; ITS: insulin-transferrin-selenium; HC: hydrocortisone; IL: GMECs are treated with an induced lactation system with EGF, ITS, and HC; IL + PRL2500/5000/7500/10,000 ng/mL: GMECs are treated with IL and 2500, 5000, 7500, or 10,000 ng/mL PRL. Data are represented as means ± SD, NS: no statistical significance, ** p < 0.01. Values with different lowercase letters indicate significant difference (p < 0.05).

Fig 4: The effect of PRL on GMECs’ lactation and SNAT1/2 expression. (A) Western blot analysis of milk protein and SNAT1/2 in GMECs treated with PRL. (B) QPCR analysis of CSN and BLG mRNA levels in GMECs treated with PRL. CSN: Casein; BLG: Beta-lactoglobulin. (C) GPO-PAP assay of TG content in the supernatant of GMECs after induction of lactation. EGF: epidermal growth factor; ITS: insulin-transferrin-selenium; HC: hydrocortisone; Control: GMECs are treated with only culture medium; IL: GMECs are treated with an induced lactation system (EGF + ITS + HC); IL + PRL2500/5000/7500/10,000 ng/mL: GMECs are treated with IL and 2500, 5000, 7500, or 10,000 ng/mL PRL. Data are represented as means ± SD. Values with different lowercase letters indicate significant difference (p < 0.05). Original images can be found in Supplementary Files.

Fig 5: PRL and SNAT1/2 levels in different stages of goats. PRL: prolactin; SNAT1/2: sodium-coupled neutral amino acid transporter 1/2. (A) Serum PRL concentrations in goats. (B) SNAT1/2 protein expressions in different stages of goat mammary tissue. Youth: The goats are in the stage of puberty; Pd91: The goats are in the stage of pregnancy, day 91; Pd137: The goats are in the stage of pregnancy, day 137; Ld4: The goats are in the stage of lactation, day 4; Ld31: The goats are in the stage of lactation, day 31. Data are represented as means ± SD. Values with different lowercase letters indicate significant difference (p < 0.05). Original images can be found in Supplementary Files.