Supplemental Material, DS1_VET_10.1177_0300985819843683 - Aging and Senescence in Canine Testes

Supplemental Material, DS1_VET_10.1177_0300985819843683 for Aging and Senescence in Canine Testes by Sophie E. Merz, Robert Klopfleisch, Angele Breithaupt and Achim D. Gruber in Veterinary Pathology</p

mCLCA3-deficiency had no impact on clinical outcome of pneumonia or bacterial loads in lungs.

<p>mClca3^−/− and WT mice were transnasally infected with 5×10⁷<i>Staphylococcus aureus</i> Newman or received PBS (controls) and body weights (A) and temperatures (B) were measured every 6 hours for 24 hours and once after 48 hours. Values are given as mean ± SEM (n = 8 each group). ^#p<0.05, ^##p<0.01, ^###p<0.001, ^####p<0.0001 versus the PBS control group. (C) Lung, liver and blood bacterial loads were determined 6 hours and 24 hours after infection. Values are given as individual data and mean (n = 11 each group). n.d.  =  not detected.</p

Early infection with <i>S. aureus</i> led to a comparable increase in systemic leukocyte response.

<p>(A, B) At indicated time points after infection, blood leukocyte numbers and subpopulations were determined by fluorescence-activated cell sorter (FACS). Values are given as mean ± SEM (n = 8 each group). ^#p<0.05, ^##p<0.01, ^###p<0.001 versus the PBS-treated control group.</p

mCLCA3-deficiency in <i>S. aureus</i> infection was not compensated by regulation of other CLCA members.

<p>Lung expression mRNA levels of mClca1 to 7 were quantified by RTq-PCR. Dotted lines indicate a fold change of 0.5 and 2, respectively, as limits for valid statement of lowered and elevated parameters. Values are given as mean ± SEM (n = 8 each group). Ct, cycle treshold.</p

Expression levels of candidate reference genes for the zebra finch song (A–C) and tissue dataset (D–H).

<p>Values are given as cycle threshold numbers (C_t values) in (A) all Area X samples combined (n = 12), (B) silent group only (n = 7), (C) singing group only (n = 5), (D) tissue dataset all samples (n = 32), (E) male brain only (n = 10), (F) female brain only (n = 10), (G) testes only (n = 6) and (H) ovaries only (n = 6).</p

Singing induced gene regulation in Area X of adult male zebra finches.

<p>Relative gene expression levels for EGR1, CFOS and FOXP2 were calculated by normalizing to the geometric mean of the two genes recommended by each of the three different algorithms. Relative expression values of the singing group are compared to the silent group. Statistical differences were calculated using the Mann Whitney U test and are indicated in the diagram (p<0.05 *, p<0.01 **, no significant difference ns).</p

Chemokine CXCL-1 and cytokine IL-17 were significantly decreased in BALF of infected mCLCA3-deficient mice.

<p>(A, B) Protein levels of cytokines were measured at 12 hours and 24 hours post infection by multiplex assay technique (IL-1β, IL-6, IL-10, IL-12p40, IL-13, CXCL-1, MCP-1, RANTES, TNF-α, GM-CSF) or by ELISA (IL-17) in BALF. (C) Expression levels of Cxcl-1, Cxcl-2 and Il-17 were quantified by RTq-PCR in the lungs. Dotted line indicates a fold change of 2 as limit for valid statement of elevated parameters. Values are given as mean ± SEM (n = 6 to 8 each group). ^#p<0.05, ^##p<0.01, ^###p<0.001, ^####p<0.0001 versus PBS control group. *p<0.05 as indicated.</p

Specificity of primers for <i>SDHA</i> in qRT-PCR of nine avian species.

<p>Melting curves of five dilutions run in triplicates are shown with one panel per species. Species names are indicated in the title of each panel.</p

Stability ranking of expression analysis for the song data set with three different statistical methods.

<p>Stability ranking of expression analysis for the song data set with three different statistical methods.</p