Fig. 1

Energy stress suppresses synthesis of milk fat and protein in HC11. A Oil red O staining images of HC11 under 0 mM glucose (left) and 10 mM glucose (right). Scale bars are 100 μm. B and C TAG concentration in cell (B) and medium (C), n = 4. 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 expression 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 of genes critical for protein synthesis. 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 Relative mRNA expressions of target proteins, n = 3. In this figure, cells for oil red O staining, TAG determination, immunofluorescence staining and WB were collected after 24 h incubation with 0 mM and 10 mM glucose mediums 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