Comparison regarding general health and behaviour between pups nursed by wild-type dams and pups nursed by α-casein deficient dams.

<p>P-values of parameters without significant differences between group 1 (G1: wild-type pups nursed by wild-type dams) and 2 (G2: wild-type pups nursed by α-casein deficient dams; P-value G1 vs. G2) and group 1 and 3 (G3: heterozygous pups nursed by wild-type dams; P-value G1 vs. G3) at 8 weeks of age (Fisher's exact test and *Mann-Whitney-U test). Constant values indicate that all animals in the groups compared had the same, normal, score.</p

Analysis of milk calcium and phosphate levels and milk protein gene expression.

<p><b>Panel A:</b> Calcium and phosphate content of mouse milk was determined as indicated in the methods section. Concentrations are given in nM. <b>Panel B:</b> Quantitative PCR analysis of α-casein and β-casein gene expression. cDNA derived from representative wild-type, heterozygous [+/−] and homozygous [−/−] α-casein deficient mice was analysed using primer pairs specific for α-casein, β-casein and the reference gene GAPDH. Expression of the casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel C:</b> Quantitative PCR analysis of γ-casein and κ-casein gene expression. Expression of the γ and κ-casein genes was correlated with the reference gene and is expressed as pg casein/pg GAPDH. <b>Panel D:</b> Correlation of casein gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Casein gene expression was correlated with the expression of the reference gene β-actin. Quantification of α-casein was done in 3 [+/+], 7 [+/−] and 5 [−/−] mice. Quantification of β-casein was done in 3 [+/+], 8 [+/−] and 4 [−/−] mice. Quantification of γ- and k-casein was done in 3 [+/+], 3 [+/−] and 3 [−/−] mice. Expression in heterozygous and α-casein deficient mice is presented as percentage of median casein gene expression in wild-type control mice [+/+] (set to 100%). Error bars represent standard deviations. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, p<0.01 by **, and p<0.001 by ***. Exact p values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021775#pone-0021775-t006" target="_blank">table 6</a>.</p

Impact of α-casein deficient milk on pup growth.

<p><b>Panel A:</b> Growth curve of three different groups of mice during lactation (G1: wild-type pups nursed by wild-type dams n = 34; G2: wild-type pups nursed by α-casein deficient [−/−] dams, n = 25; and G3: heterozygous pups nursed by wild-type dams, n = 22). Values shown are +/− standard deviation. All weight differences between group G2 vs. G1 and G3 were significant from day 7 (p<0.001) as assessed by ANOVA. <b>Panel B:</b> Percentage weight gain throughout different stages of life for the three experimental groups. The weight of individual mice was compared on two days (as indicated: e.g. 1/3 corresponds to the interval between day 3 and day 1 of life) and the percent weight increase was recorded. The average for all mice in the three experimental groups is shown for consecutive time periods. <b>Panel C:</b> Growth curve of mice in the three groups over the first 6 months of life. Mice nursed by α-casein deficient dams show a consistent growth deficiency. <b>Panel D:</b> Growth curve of mice in the three groups of mice over the first 6 months of life separated by gender. Error bars represent standard deviations.</p

Analysis of markers of apoptosis in mammary tissue from α-casein deficient mice.

<p><b>Panel A:</b> Western blot analysis of samples derived from two α-casein deficient mice and one heterozygous mouse (all taken at mid-lactation). The protein extracts were separated on a 10% (upper panel) and 15% (lower panel) polyacrylamide-gel blotted to nitrocellulose and detected using antisera against β-actin (upper panel) and the cleavage product of caspase 3 (lower panel). Extracts from RAW264 cells treated with 10 µM staurosporin (STS) for 6 h were used as positive control. The sizes of the protein molecular weight markers (Cell Signaling Technologies, biotinylated protein marker) are indicated as are the positions of the β-actin and caspase 3 proteins (arrows) <b>Panel B:</b> Analysis of caspase 3 and caspase 7 activity in cytoplasmic extracts of mammary gland tissue of control [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using a Caspase-Glo assay (Promega). Extracts derived from RAW264 cells treated with staurosporin were used as positive control. <b>Panel C:</b> Correlation of gene expression in wild type [+/+], heterozygous [+/−] and α-casein deficient mice [−/−] using quantitative PCR. Expression of the genes encoding the apoptosis related proteins nucleolar protein 3 (Nol3; up-regulated), Birc5 (up-regulated) and Traf1 (down-regulated) were correlated with the expression of the reference gene β-actin. Quantification was done in 3 [+/+], 6 [+/−] and 5 [−/−] mice. Statistical analysis using one-way ANOVA demonstrates that the expression changes for all three genes observed in α-casein deficient mice with respect to both wild-type and heterozygous mice occur with p<0.05. For comparisons against wild-type mice in a one-way ANOVA p<0.05 is indicated by *, and p<0.001 by ***.</p

Immuno-histochemistry analysis of mammary tissue.

<p><b>Panel A:</b> Paraffin embedded sections of mammary tissue (day 10 lactation) were analysed using a rabbit-anti α-casein antiserum. The slides were subsequently counterstained with haematoxylin. Representative sections derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice are presented. The lower panels are control sections incubated with a rabbit pre-immune serum in place of the α-casein specific antiserum. <b>Panel B:</b> Representative sections derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice at day 10 of lactation stained with haematoxylin.</p

Significance of changes in protein ratios.

<p>Milk protein expression was analysed in wild-type mice [+/+] (n = 3), heterozygous mice [+/−] (n = 6) and α-casein deficient mice [−/−] (n = 5). Expression was quantified by densitometric scanning of protein gels and correlation of the net intensities with a molecular weight marker of known concentration. The protein ratios of α-casein to albumin (acas/alb.), α-casein to β-casein (acas/bcas), β-casein to γ-casein (bcas/gcas), β-casein to albumin (bcas/alb.) and γ-casein to albumin (gcas/alb.) were determined. The data were analysed by ANOVA for one way comparisons. The p values obtained for the different comparisons are shown. P values in bold print are below the cut off points of 0.01 or 0.001 (as indicated).</p

Experimental groups.

<p>Experimental groups.</p

Analysis of milk protein expression.

<p><b>Panel A:</b> Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The α-casein protein was detected using a rabbit-anti α-casein antiserum. <b>Panel B:</b> Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The β-casein protein was detected using a goat-anti β-casein antiserum. <b>Panel C:</b> Western blot analysis of milk derived from wild-type [+/+], heterozygous [+/−] and homozygous [−/−] α-casein deficient mice. The grp78/BiP protein was detected using a goat-anti grp78 antiserum. <b>Panel D:</b> Densitometric analysis of milk protein abundance as detected by SDS-PAGE. Coomassie Blue stained gels with milk samples from wild-type [+/+] (n = 3), heterozygous [+/−] (n = 6) and homozygous [−/−] α-casein deficient mice (n = 5) were scanned in a Kodak densitometer and the net intensity of the milk proteins was compared with the intensities of molecular weight marker proteins of know concentration. Protein concentrations detected in the three genotypes for albumin, α-casein (acas), β-casein (bcas) and γ-casein protein (gcas) are presented. Comparisons of [−/−] vs [+/−] as analysed by one-way ANOVA are significant with p<0.001 (***); Comparisons of [+/−] vs [+/+] are significant with p<0.05 (*) where indicated. <b>Panel E:</b> The relative amounts of milk protein abundance were calculated for the ratios of α-casein to albumin (acas/alb.), α-casein to β-casein (acas/bcas), β-casein to γ-casein (bcas/gcas), β-casein to albumin (bcas/alb.) and γ-casein to albumin (gcas/alb.). For comparisons against wild-type mice in a one-way ANOVA p<0.01 is indicated by **, p<0.001 by ***. Exact P values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021775#pone-0021775-t005" target="_blank">table 5</a>.</p

Behavioural differences between pups nursed by wild-type dams and pups nursed by α-casein deficient dams during the lactation period; days of assessment: 1, 3, 7, 14 and 21.

<p>Significance level p<0.05 for Fisher's exact test. Y = yes, n = no, ns = not significant, norm = normal, consp = conspicuous.</p

Significance of changes in casein gene expression.

<p>Casein gene expression was measured in wild-type mice [+/+], heterozygous mice [+/−] and α-casein deficient mice [−/−] using quantitative PCR. The results were correlated with the expression of the reference gene β-actin. Quantification of α-casein was done in 3 [+/+], 7 [+/−] and 5 [−/−] mice. Quantification of β-casein was done in 3 [+/+], 8 [+/−] and 4 [−/−] mice. Quantification of γ- and k-casein was done in 3 [+/+], 3 [+/−] and 3 [−/−] mice. The data were analysed by ANOVA for one way comparisons. The p values obtained for the different comparisons are shown. P values in bold print are below the cut off points of 0.01 or 0.001 (as indicated).</p