Fig. 2

AMPK activation attenuates 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 cells (B) and medium (C), n = 4. D Heatmap analysis of up-regulated (red) or down-regulated genes (green) in HC11, n = 3. The 0 mM glucose group and the Phe group were compared with the 10 mM group respectively, * means P < 0.05, ** means P < 0.01 and *** means P < 0.001. E–G Western blots and real time PCR of HC11 lysates for critical fat synthesis genes. E Representative western-blotting bands hybridized with target proteins including FASN, ACACA, FABP3, DGAT1, SREBP1 and Actin antibodies, n = 3. (F) is the result of analyzing the WB bands and (G) represents data from real-time PCR, n = 3. H 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. I–K Western blots and real time PCR of HC11 lysates for critical protein synthesis genes. I Representative western-blotting bands hybridized with target proteins including α-casein, β-casein, WAP and Actin antibodies, n = 3. J Result of analyzing the WB bands and (K) represents data from real-time PCR, n = 3. In this figure, cells for oil red O staining, TAG determination, lipidomics, immunofluorescence staining and WB were collected after 24 h incubation with 0 mM glucose medium, 10 mM glucose medium, (10 mM glucose + 1.5 mM AICAR) medium and (10 mM glucose + 300 μM Phe) medium, and cells for real-time PCR are 12 h. All data 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