Effect of AN-7 on the activity and expression of cellular HDACs classes I and II.

<p>(A) The cells were exposed to AN-7 for 3 h in the presence of the HDAC fluorogenic substrate. The average % of HDAC activity as a function of 0–100 µM concentration of AN-7 of a representative experiment conducted in quadruplicates is shown. (B) The average IC₅₀±SE of AN-7 for each of the cell types is calculated from three independent experiments. (C) Lysates of cells were loaded (35 µg protein/well) on 10% SDS gels and were subjected to Western blot analysis. HDAC1, HDAC2, HDAC5 or actin were detected with the appropriate antibodies. (D) HDAC activity of three experiments (in triplicate) conducted with 4T1 (cancer) and H9C2 (non-cancer) cells treated with AN-7 (50 µM) as a single agent for a total of 3 h, 1 h prior to the addition of the fluorogenic substrate and 2 h in its presence; Dox (200 nM) as a single agent treatment, or with the combination AN-7+Dox where 50 µM AN-7 was added 1 h prior to the addition of Dox and the fluorogenic substrate and then incubated for an additional 2 h. (E) The viability of the cells treated as described in (D) was determined by a Hoechst assay after 23 or 24 h for cells treated with Dox. (F) Cells were treated with AN-7 (3 or 24 h), Dox (2 or 23 h) or AN-7 (3 or 24 h)+Dox (2 or 23 h), as described (D). The lysates of the cells were subjected to Western blot analysis as described (C).</p

Effect of AN-7, DOX and their combinations on angiogenesis.

<p>Sections of tumors and hearts were stained for (A) lo-FGF (DAB); (B) VEGF (Nova Red) and counter stained with hematoxylin. Bar = 200 µm, the 4-fold magnified picture in the insert was taken from the area indicated by the square. (C) Lysates samples (as described above) were resolved on 7.5% SDS gels and stained for TSP-1(left). Mean±SE of the ratio of TSP-1/actin expression is shown (right), ^#p<0.05, as specified, vs. untreated control; * p<0.05, as specified, vs. Dox; ^&p<0.05, AN-7+Dox vs. AN-7.</p

Effect of AN-7, DOX and their combinations on markers of proliferation and fibrosis.

<p>Sections of tumors and hearts were stained (DAB) for: (A) Ki-67; (B) c-Kit and both were counter stained with hematoxylin. The arrowhead marked the c-Kit positively staind cells. Bar = 200 µm, the 4-fold magnified picture in the insert was taken from the area indicated by the square. (C) Lysates samples, as described above, were resolved on 10% SDS gel and stained for c-Myc (left). The Mean±SE ratio of c-Myc/actin expression is shown (right). ^#p<0.05, as specified, vs. untreated control; *p<0.05, as specified, vs Dox. (D) Picrosirius red and fast-green for the visualization of interstitial fibrosis. Bar = 200 µm, the 2-fold magnified picture in the insert was taken from the area indicated by the square.</p

Effect of Dox, AN-7, and AN-7+Dox in the 4T1 murine mammary carcinoma model.

<p>(A) In the preliminary experiment, female Balb/c mice carrying 75–180 mm³ subcutaneous 4T1 tumors were randomly divided into three groups (10 mice/group) and were injected ip once a week with either vehicle, 3 or 5 mg/kg Dox. Tumor volume was measured twice a week up to day 14 when the experiment was terminated (after 2 Dox treatments). Mean±SE of tumor volume was: *p<0.05, untreated vs. the other groups; #p<0.05, Dox 5 mg/kg vs. Dox 3 mg/kg. (B) Mean±SE of tumor weight and the ratio of heart-weight to body-weight (HW/BW, g/g), measured at the experiment termination point were: *p<0.01, Dox 5 mg/kg vs. all other groups. (C) In the pivotal experiment, the Percent Failure Free mice bearing 75–180 mm³ subcutaneous 4T1 tumors, was assessed by a Kaplan-Mier graph. The mice were assigned to the following treatments: saline vehicle ip (n = 7) or po (n = 7); ip Dox, 5 mg/kg once/week (n = 14); po AN-7, 50 mg/kg 3 times/week (n = 14); po AN-7+ip Dox (n = 14). The arrows on the x-axis indicate the point in time of the second and the third Dox treatment. (D) Mean±SE of heart-weight to body-weight ratios (HW/BW, g/g) at the above experiment end-point was ^#p<0.01, Dox 5 mg/kg vs. all other groups. (E) Tumor weight at the termination point (mean±SE) was: Dox 5 (total) represents the tumor weight of all the mice treated with 5 mg/kg Dox (n = 14), as measured at their individual end-points; Dox 5 (>21) represents the tumor weight of the mice (n = 5) treated with 5 mg/kg Dox and survived beyond day 21 of treatment. *p<0.02 all groups vs. untreated control; ^#p<0.05 AN-7+Dox vs. AN-7 or Dox 5 (total) or p<0.01 Dox 5 (>21). (F) Mean±SE of lung lesions for the various treatment groups is shown. *p<0.02, untreated vs. all treatment groups; ^#p<0.05, AN-7+Dox vs. AN-7 or vs. Dox.</p

Effect of AN-7, DOX and AN-7+Dox on HDAC1 and HDAC2 expression in tumor or heart.

<p>(A) Extracts of tumors from 8 mice and hearts from 6 mice per treatment group were loaded (20 µg protein/well) on 10% SDS gels, and subjected to Western blot analysis. HDAC1, HDAC2 or actin were detected with the appropriate antibodies. (B) Expression of HDAC1, HDAC2 or actin in tumors and hearts was analyzed by Odyssey 2.1. The intensity ratio of HDACs to actin (mean±SE) is presented. *p<0.05, tumors of untreated or Dox-treated vs. AN-7 or AN-7+Dox; ^#p<0.007, HDAC2 expression in the hearts of Dox treated vs. all other treatment groups.</p

Protein expression (IMH) of VIP, VPAC1-R and VPAC2-R in human ovaries.

<p><u>Note</u>: IMH = immunohistochemistry, GW = gestational weeks. Percentages represent the proportion of the samples with follicular staining.</p><p>Staining intensities: + = positive staining; full/partial = full or partial cytoplasmic staining.</p

IMH photographs of VIP protein expression.

<p>(A) Section of human ovary from a 22-GW-old fetus. Note the primordial follicles, weak red-brown staining indicating VIP expression in oocytes (full cytoplasmic and nuclear staining), and weak staining in a portion of the GC and stroma cells. Original magnification X400. (B) Section of human ovary from a 6-year-old girl. Note the primordial follicles with red-brown staining indicating VIP protein expression in the oocyte (mainly cytoplasmic staining and nuclear staining), and in a portion of the GC and a portion of the stroma cells. Original magnification X400. (C) Positive control for VIP protein expression of section of mouse brain. Note the red-brown staining in the sample. Original magnification X400. (D) Negative control for the same ovarian section as in panel A. Note the primordial follicles with the overall blue staining and the lack of red-brown staining. Original magnification X400. (E) Negative control for the same ovarian section as in panel B. Note the primordial follicles with overall blue staining and the lack of red-brown staining. Original magnification X400.</p

IMH photographs of VPAC2-R protein expression.

<p>(A) Section of a human ovary from the same fetus as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037015#pone-0037015-g001" target="_blank">Figure 1</a> (A) and (D). Note the red-brown staining indicating VPAC2-R expression in the oocytes (full weak cytoplasmic staining with nuclear staining), in a portion of the GC and stroma cells. Original magnification X400. (B) Section of human ovary from the same woman as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037015#pone-0037015-g002" target="_blank">Figure 2</a> (A) and (B). Note the secondary follicle (full cytoplasmic staining with nuclear staining), with red-brown staining in the GC and stroma cells. Original magnification X400. (C) Negative control for the same ovarian section as in panel A. Note the primordial follicles with overall blue staining and lack of red-brown staining. Original magnification X400. (D) Negative control for the same ovarian section as in panel B. Note the primordial follicles with overall blue staining and lack of red-brown staining. Original magnification X400.</p

IMH photographs of VPAC1-R protein expression.

<p>(A) Section of human ovary from a 22-year-old woman. Note the primordial follicles, with red-brown staining indicating VPAC1-R expression in the oocytes (full cytoplasmic staining and nuclear staining), and in a portion of the GC and stroma cells. Original magnification X400. (B) Negative control for the same ovarian section as in panel A. Note the primordial follicle, overall blue staining, and lack of red-brown staining. Original magnification X400.</p

Representative RT-PCR gel illustrating expression of the VIP, VPAC1-R and VPAC2-R genes in fetal and adult ovaries.

<p>(A) VIP gene (134 base pairs), (B) VPAC1-R gene (97 base pairs), (C) VPAC2-R gene (155 base pairs), (D) DHFR gene as positive control (231base pairs). Sample 1: Ovary from a 21-year-old woman, Sample 2: Ovary from a 29-year-old woman, Sample 3: Ovary from a 39-year-old woman, Sample 4: Ovary from a 21-GW-old fetus, Sample 5: Ovary from a 23-GW-old fetus, Sample 6: Ovary from a 25-GW-old fetus, Sample 7: Ovary from a 27-GW-old fetus. M: Marker (100 base pair DNA ladder, QIAGEN), +: RT presence, -: RT absence.</p