Fig. 3

Inhibition of AMPK promotes the synthesis of milk fat and protein in HC11. A Oil red O staining images of HC11. Scale bars are 100 μm. B and C TAG concentration in cell (B) and medium (C), n = 6. D–F Western blots of HC11 lysates. In (D), bands were incubated with target proteins including FASN, ACACA, FABP3, DGAT1, SREBP1 and Actin antibodies, n = 3. Protein expressions of target proteins are shown in (E), n = 3. F Relative mRNA expressions of target proteins, n = 3. G Confocal microscopy of HC11 immunostained for α-casein (green), β-casein (green), WAP (green) and DAPI (blue). Scale bars, 100 μm. The small red boxes in the figure are typical areas selected, and the three small images on the right are the enlarged images of the target protein, nucleus and merge images in selected areas, respectively. H–J Western blots and real time PCR of HC11 lysates for critical protein synthesis genes. In (H), bands were incubated with target proteins including α-casein, β-casein, WAP and Actin antibodies, n = 3. Protein expressions of target proteins are shown in (I), n = 3. (J) shows relative mRNA expressions of target proteins, n = 3. K TAG concentration in cell (top) and medium (bottom), n = 6. Cells are first transfected with siRNA and treated with the corresponding medium, the cell lysate and medium are then collected for detection. L Western blots of HC11 lysates for AMPK signaling pathway, critical milk protein and fat synthesis genes. In this figure, cells for oil red O staining, TAG determination, immunofluorescence staining and WB were collected after 24 h incubation with 0 mM glucose medium, 2 mM glucose medium and (2 mM glucose + 50 μM Compound C) medium and cells for real-time PCR are 12 h. All datas with error bars are averages ± SEM. In histograms, the numbers above the column are the p-values marked as a range if significantly different (P < 0.05) and each small black dot represents a test value