Changes in expression levels of genes that are identified as target genes for the transcription factor Ace2 in the <i>med5/med15</i>, <i>med15/med16</i> and <i>med15</i> Degron strains relative to wild type cells.

<p>Changes in expression levels of genes that are identified as target genes for the transcription factor Ace2 in the <i>med5/med15</i>, <i>med15/med16</i> and <i>med15</i> Degron strains relative to wild type cells.</p

Flow cytometry analyses of Degron constructs.

<p>DNA content of cells carrying the indicated Degron constructs was analyzed by flow cytometry at 3 hours after switching from the permissive to the restrictive growth conditions. Numbers below each histogram indicate the percentage of cells in the G1-, the S-, and the G2+M-phases, respectively.</p

Genes whose expression is changed 1.5-fold or more in the <i>med5/med15</i> double Degron strain compared to either of the wild type, <i>med5</i> or <i>med 15</i> and in the <i>med15/med16</i> double Degron strains compared to either of the wild type, <i>med15</i> or <i>med 16</i> strain grown at the restrictive conditions.

<p>Genes whose expression is changed 1.5-fold or more in the <i>med5/med15</i> double Degron strain compared to either of the wild type, <i>med5</i> or <i>med 15</i> and in the <i>med15/med16</i> double Degron strains compared to either of the wild type, <i>med15</i> or <i>med 16</i> strain grown at the restrictive conditions.</p

Transcription profile analysis using AffymetrixsYeast Genome 2.0 Array.

<p>25 genes were differently regulated in both of the double-Degron strains (<i>med5/med15</i> (A) and <i>med15/med16</i> (B)), 45 minutes after induction of degradation, compared to the single Degron (<i>med5</i>, <i>med15</i> and <i>med16</i>) and wild type (Wt) strains, as shown in the Venn diagrams (FDR<0.05 and FC>abs(log2(1.5)).</p

Confirmation of Ace2 target genes.

<p>mRNA levels of the genes CTS1, EXG1 and YHB1 from WT and Degron-strains were measured using qPCR and normalized against the WT level. qPCR levels are compared to the levels determined in the corresponding microarray assays. The experiments were performed in biological triplicates, and error bars represent the standard deviation. P-values where calculated using Student’s t-test. * Indicates p-value < 0.05, ** indicates p-value <0.01.</p

Double deletions of yeast <i>MED5/MED15</i> and <i>MED15/MED16</i> are synthetically lethal.

<p>(A) The strains indicated to the left were plated as 10-fold serial dilutions, 5 µl spots at permissive conditions (24°C, Cu⁺⁺, glucose) and restrictive conditions (37°C, no Cu⁺⁺, galactose) respectively. (B) The strains indicated to the right were grown in liquid media, at restrictive conditions, for 24 hours. OD₆₀₀ was measured at the indicated time points. OD₆₀₀ at 0 hours was set to 1 and the other values where normalized against time = 0 min. The graphs represent the mean of two separate experiments. The graphs represent the mean of two separate experiments, and the error bars represent the standard deviation.</p

Composition of sublingual films of biochanin A.

<p>*Quantities are expressed in %w/w of polymer; **Quantities are expressed in ratio of biochanin A to excipient.</p><p>Composition of sublingual films of biochanin A.</p

IC₅₀ values (µM) of isoflavones on <i>C. pneumoniae</i> and <i>C. trachomatis</i> inclusion counts.

<p>*IC₅₀ values were determined by treating the infected cell cultures with isoflavones at concentration 100, 75, 50, 25, 10 and 5 µM and determining the inclusion counts as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115115#s2" target="_blank">Materials and methods</a>. -  =  no inhibition. A statistically significant difference in <i>C</i>. <i>pneumoniae</i> IC₅₀ values was observed between biochanin A and genistein (p<0.05, Student's t-test)</p><p>IC₅₀ values (µM) of isoflavones on <i>C. pneumoniae</i> and <i>C. trachomatis</i> inclusion counts.</p

Effect of isoflavones on <i>C. trachomatis</i> inclusion size.

<p>A) Immunofluorescence images of <i>C. trachomatis</i> infected HeLa cells (untreated and treated with 100 or 10 µM biochanin A). <i>Chlamydia</i> inclusions are stained in green (polyclonal rabbit antibody raised against formalin fixed <i>C</i>. <i>trachomatis</i> elementary bodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115115#pone.0115115-Marwaha1" target="_blank">[30]</a>) and host cell nuclei are stained in blue with DAPI. B) Quantitation of average inclusion sizes in <i>C. trachomatis</i> infections treated with the isoflavones. Inclusion sizes are expressed as relative units proportional to the untreated controls. In a pairwise comparison of different concentrations, the mean inclusion size in biochanin A, genistein and genistin treated samples were statistically significantly smaller than in formononetin, daidzein and daidzin treated samples, respectively (p<0.05, Student's t-test).</p

X-ray diffraction and DSC measurements of film formulations.

<p>A) X-ray diffraction patterns of (a) biochanin A powder, (b) genistein powder, (c) HPMC powder, (d) film Fii, (e) blank film Faa without biochanin A, (f) film Faa, (g) film Fjj and (h) physical mixture of formulation Faa. B) DSC thermograms of (a) biochanin A powder, (b) HPMC powder, (c) film Fii, (d) blank film Faa without biochanin A, (e) film Faa, (f) film Fjj and (g) physical mixture of formulation Faa.</p