Journal: Journal of Animal and Feed Sciences

Article Title: Sodium selenite addition promotes the proliferation of bovine mammary epithelial cells through the Akt-mTOR signalling pathway

doi: 10.22358/jafs/159458/2023

Figure Lengend Snippet: Figure 1. Effect of sodium selenite (Na2SeO3) on the proliferation of bovine mammary gland epithelial cells (BMECs). (A) Effect of different doses of Na2SeO3 (0, 9, 18, 35, 70, 140, 280 and 560 nM) on the proliferation of BMECs after 3-day culture as determined by the Cell Counting Kit 8 (CCK-8) assay. (B) Effect of Na2SeO3 (0 and 35 nM) on BMEC proliferation as determined by the EdU incorporation assay. (C) Analysis of EdU-positive cell percentage in panel B. (D) mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2) and cyclin D1 (CCND1) in re- sponse to 35 nM Na2SeO3. (E) Western blot analysis of PCNA and Cyclin A1 proliferation markers in BMECs after 3-day culture; β-actin was used as a loading control. (F) Mean ± SEM (standard error of the mean) of immunoblotting bands of PCNA and Cyclin A1, and band intensities are expressed in arbitrary units. Bars that do not share the same capital letter are significantly different (P < 0.01). * P < 0.05 and ** P < 0.01 compared to the control group.

Article Snippet: Cyclin A1 was targeted using a mouse anti-cyclin A1 monoclonal antibody (Cat. no. NB100-2660; Novus Biologicals LLC, Centennial, CO, USA), proliferating cell nuclear antigen (PCNA) was targeted using a mouse anti-PCNA monoclonal antibody (Cat. no. 2586; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt and β-actin were targeted with rabbit monoclonal antibodies against Akt and β-actin (Cat. no. 9272 and 4970; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated p-AktSer473, p-mTORSer2448 and mTOR were targeted with rabbit polyclonal antibodies against p-AktSer473, p-mTORSer2448 and mTOR (Cat. no. bs-0876R, 3495R and 1992R; BIOSS, Beijing, China), B-cell lymphoma 2 (BCL2), Caspase-3 and Caspase-9 were targeted with rabbit polyclonal antibodies against BCL2, Caspase-3 and Caspase-9 (Cat. no. bs-20351R, 0081R and 0049R; BIOSS, Beijing, China), BCL2-associated X (BAX) was targeted with a mouse anti-BAX polyclonal antibody (Cat. no. bs-0127M; BIOSS, Beijing, China); goat anti-rabbit (Cat. no. bs-0295G) and goat anti-mouse (Cat. no. bs0296G) secondary antibodies were purchased from BIOSS (Beijing, China).

Techniques: Cell Counting, CCK-8 Assay, Expressing, Western Blot, Control

Journal: Journal of Animal and Feed Sciences

Article Title: Sodium selenite addition promotes the proliferation of bovine mammary epithelial cells through the Akt-mTOR signalling pathway

doi: 10.22358/jafs/159458/2023

Figure Lengend Snippet: Figure 4. Suppression of Akt reversed the stimulation of bovine mammary gland epithelial cell (BMEC) proliferation and activation of the mTOR signalling pathway modulated by 35 nM sodium selenite (Na2SeO3). (A) Effect of 30 nM Akt inhibitor-1 (AKT-IN-1) on the proliferation of BMECs after 3-day incubation was determined using the Cell Counting Kit 8 (CCK-8) assay. (B) mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2) and cyclin D1 (CCND1), B cell lymphoma 2 (BCL2), BCL2-associated X 4 (BAX4),BCL2/BAX4, caspase 3 (CASP3) and caspase 9 (CASP9) in response to 35 nM Na2SeO3 and/or 30 nM AKT-IN-1. (C) Western blot analysis of PCNA, Cyclin A1, BCL2, BAX, BCL2/BAX, Caspase-3, Caspase-9, p-Akt, Akt, p-mTOR and mTOR in BMECs after 3-day culture in the presence of 35 nM Na2SeO3 and/ or 30 nM AKT-IN-1; β-actin was used as a loading control. (D) Mean ± SEM (standard error of the mean) of immunoblotting bands of PCNA, Cyclin A1, BCL2, BAX, BCL2/BAX, Caspase-3, Caspase-9, p-Akt/Akt and p-mTOR/mTOR, and band intensities are expressed in arbitrary units. * P < 0.05 and ** P < 0.01 compared to the control group, # P < 0.05 and ## P < 0.01 compared to the Na2SeO3 group.

Article Snippet: Cyclin A1 was targeted using a mouse anti-cyclin A1 monoclonal antibody (Cat. no. NB100-2660; Novus Biologicals LLC, Centennial, CO, USA), proliferating cell nuclear antigen (PCNA) was targeted using a mouse anti-PCNA monoclonal antibody (Cat. no. 2586; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt and β-actin were targeted with rabbit monoclonal antibodies against Akt and β-actin (Cat. no. 9272 and 4970; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated p-AktSer473, p-mTORSer2448 and mTOR were targeted with rabbit polyclonal antibodies against p-AktSer473, p-mTORSer2448 and mTOR (Cat. no. bs-0876R, 3495R and 1992R; BIOSS, Beijing, China), B-cell lymphoma 2 (BCL2), Caspase-3 and Caspase-9 were targeted with rabbit polyclonal antibodies against BCL2, Caspase-3 and Caspase-9 (Cat. no. bs-20351R, 0081R and 0049R; BIOSS, Beijing, China), BCL2-associated X (BAX) was targeted with a mouse anti-BAX polyclonal antibody (Cat. no. bs-0127M; BIOSS, Beijing, China); goat anti-rabbit (Cat. no. bs-0295G) and goat anti-mouse (Cat. no. bs0296G) secondary antibodies were purchased from BIOSS (Beijing, China).

Techniques: Activation Assay, Incubation, Cell Counting, CCK-8 Assay, Expressing, Western Blot, Control

Journal: Journal of Animal and Feed Sciences

Article Title: Sodium selenite addition promotes the proliferation of bovine mammary epithelial cells through the Akt-mTOR signalling pathway

doi: 10.22358/jafs/159458/2023

Figure Lengend Snippet: Figure 5. Suppression of the mTOR signalling pathway eliminated the promotion of bovine mammary gland epithelial cell (BMEC) proliferation modulated by 35 nM sodium selenite (Na2SeO3). (A) Effect of 50 pM rapamycin (Rap), an inhibitor of mTOR, on the proliferation of BMECs after 3-day incubation was determined using the Cell Counting Kit 8 (CCK-8) assay. (B) mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin A2 (CCNA2) and cyclin D1 (CCND1), B cell lymphoma 2 (BCL2), BCL2-associated X 4 (BAX4), BCL2/BAX4, caspase 3 (CASP3) and caspase 9 (CASP9) in response to 35 nM Na2SeO3 and/or 50 pM Rap. (C) Western blot analysis of PCNA, Cyclin A1, BCL2, BAX, BCL2/ BAX, caspase-3, caspase-9, p-Akt, Akt, p-mTOR and mTOR in BMECs after 3-day culture in the presence of 35 nM Na2SeO3 and/or 50 pM Rap; β-actin was used as a loading control. (D) Mean ± SEM (standard error of the mean) of immunoblotting bands of PCNA, Cyclin A1, BCL2, BAX, BCL2/BAX, Caspase-3, Caspase-9, p-Akt/Akt and p-mTOR/mTOR, and band intensities are expressed in arbitrary units; * P < 0.05 and ** P < 0.01 compared to the control group, # P < 0.05 and ## P < 0.01 compared to the Na2SeO3 group.

Article Snippet: Cyclin A1 was targeted using a mouse anti-cyclin A1 monoclonal antibody (Cat. no. NB100-2660; Novus Biologicals LLC, Centennial, CO, USA), proliferating cell nuclear antigen (PCNA) was targeted using a mouse anti-PCNA monoclonal antibody (Cat. no. 2586; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt and β-actin were targeted with rabbit monoclonal antibodies against Akt and β-actin (Cat. no. 9272 and 4970; Cell Signaling Technology, Inc., Danvers, MA, USA), phosphorylated p-AktSer473, p-mTORSer2448 and mTOR were targeted with rabbit polyclonal antibodies against p-AktSer473, p-mTORSer2448 and mTOR (Cat. no. bs-0876R, 3495R and 1992R; BIOSS, Beijing, China), B-cell lymphoma 2 (BCL2), Caspase-3 and Caspase-9 were targeted with rabbit polyclonal antibodies against BCL2, Caspase-3 and Caspase-9 (Cat. no. bs-20351R, 0081R and 0049R; BIOSS, Beijing, China), BCL2-associated X (BAX) was targeted with a mouse anti-BAX polyclonal antibody (Cat. no. bs-0127M; BIOSS, Beijing, China); goat anti-rabbit (Cat. no. bs-0295G) and goat anti-mouse (Cat. no. bs0296G) secondary antibodies were purchased from BIOSS (Beijing, China).

Techniques: Incubation, Cell Counting, CCK-8 Assay, Expressing, Western Blot, Control