Cell viability with various centrifugation rates (400, 1500, 3000, 5000×<i>g</i> during 10 min at 4°C) comparing to the reference milk cell suspension without supplementary centrifugation (Ref).

<p>(A) Boxplot and whiskers of flow cytometry results with milk somatic cell suspensions incubated with specific antibodies and vioblue live/dead kit (n = 4); (B) Cell viability of each type cell for 400×<i>g</i> (white bars), 1500×<i>g</i> (hatched bars), 3000×<i>g</i> (grey bars), 5000×<i>g</i> (dark bars) centrifugations by flow cytometry; (C) Mean proportion of macrophages (dark sectors), PMNs (grey sectors) and lymphocytes (white sectors) in cell samples under various centrifugation with different gravitational velocities. Means with different superscripts (a-d) differ significantly (<i>P</i><0.05).</p

Summary of the experimental design of the somatic cell preparation and the various treatments applied, i.e. storage in milk or PBS for 72 h, variation of the centrifugation rates and heat treatment.

<p>Summary of the experimental design of the somatic cell preparation and the various treatments applied, i.e. storage in milk or PBS for 72 h, variation of the centrifugation rates and heat treatment.</p

Flow cytometry identification of differential somatic cell count and simultaneous their quantification of all cells and each cell type.

<p>(A)(B)(C) fresh blood cell suspension, (D)(E)(F) milk cell suspension after 80°C × 30 min and (G)(H)(I)milk cell suspension conserved at 4°C in PBS pH 7.4. The cell subpopulations of each sample were shown in APC/FITC dotplot (A)(D)(G), the viable and non-viable population of all cells and each subpopulation were shown in histogram-Vioblue scatter (B)(E)(H) and (C)(F)(I), respectively.</p

Flow cytometry scatter pattern for the identification of differential somatic cells in blood-milk mix cell suspension (1/1, v/v) and corresponding isotype control sample, respectively.

<p>(A) FSC/SSC dotplot of cell suspension. All somatic cells (in yellow) in cell suspension (B) were identified by CD45/PerCp+. The subpopulation of cell suspension in APC/FITC dotplot (C), macrophages (red), PMNs (blue) and lymphocytes (green) are identified by CD14/APC+ gate in APC/SSC plot (D), CH138A/FITC+ gate in FITC/SSC plot (E), and FSC/SSC size/granularity gate (F), respectively.</p

Evaluation of transcripts in the bovine mammary gland during pregnancy using RNA-Seq analyses.

<p>Evaluation of transcripts in the bovine mammary gland during pregnancy using RNA-Seq analyses.</p

Description of the bovine <i>CSN</i> gene cluster and the distal upstream region of <i>CSN1S1</i>.

<p><b>(A)</b> The bovine <i>CSN</i> gene cluster, located on BT6 from position 87,140 kb to 87,395 kb (Ensembl release 75, February 2014), is shown at the top of the Figure. Transcripts are indicated below. <b>(B)</b> Schematic representation of the bovine <i>CSN1S1</i> gene and its upstream region on BT6. The transcription start point (tsp, +1) of the bovine <i>CSN1S1</i> gene is located at position 87,141,591 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111556#pone.0111556-Stewart1" target="_blank">[34]</a>. The distal upstream regulatory region depicted in this Figure is located between 87,131,271 and 87,131,750. It extends from 10,320 to 9,841 nt upstream from the tsp. The <i>CSN1S1</i> coding exons are depicted by black boxes, and the 3′ and 5′ untranslated regions by white boxes. Two distal potential STAT5 binding sequences (D1 and D2) are indicated by white hexagons, the proximal consensus STAT5 binding site (C) is indicated by a grey hexagon. The four CpG are indicated by lollipops. <b>(C)</b> Sequence of the <i>CSN1S1</i> distal upstream region depicted in (B). Sequences which were used to design PCR primers or pyrosequencing primers are italicized and underlined or are in bold and highlighted in grey, respectively. Their orientation is indicated by arrows. The four C studied (within the CpG sequence) located at −10276, −10216, −10196 and −9919 from the tsp previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111556#pone.0111556-Stewart1" target="_blank">[34]</a> and at positions 87.131.315; 87.131.375; 87.131.395 and 87.131.672 on BT6 are indicated in bold and circled. The two potential STAT5 binding sites from −9929 to −9910 and from −9907 to −9888 are boxed.</p

Primers used to analyse RNA transcribed from the CSN1S1 upstream region.

<p>Primers used to analyse RNA transcribed from the CSN1S1 upstream region.</p

Evaluation of transcripts in the bovine mammary gland during lactation after ODM or TDM using RT-qPCR analyses.

<p>Evaluation of transcripts in the bovine mammary gland during lactation after ODM or TDM using RT-qPCR analyses.</p

Average milk yield for each udder half of the eight cows during the three periods.

<p>Milk yield from the left right udder is indicated by black squares, whereas that of the left one is indicated by grey diamonds. The three periods are: P1, twice daily milking of both udder halves; P2, twice daily milking of the right udder half and once daily milking of the left one; P3, twice daily milking of both udder halves. Significant differences between the amounts of milk produced by the two udder halves are indicated by stars: *** P<0.001; * P<0.05.</p

Description of sense probes used in EMSA.

<p>Description of sense probes used in EMSA.</p