Immunohistochemical analyses revealed RFC40, PCNA, Cyclin A, phosphor-Aurora A/B/C kinase and pHis3 positive CMs nuclei in hypertrophied RV.

<p>Control (Con) and SUHxNx-5 wks (5 wks) RV sections (n = 3) were incubated with polyclonal anti-RFC40 (A) anti-PCNA (B), anti-Cyclin A (C), anti-phosho Aurora A/B/C kinase (p-Aurora-A/B/C; D) and anti-H3P (E) antibodies, respectively, and then with Alexa-568-labeled anti-rabbit and Alex-488-labeled anti-mouse secondary antibodies, respectively. Images were collected using an Olympus Plan x20/NA 0.25 Phi objective. In each experiment, all data were collected at identical imaging settings. Arrows indicates positive CMs.</p

Chromosomal missegregation/Aneuploidy was observed in the CM nuclei from the hypertrophied RVs.

<p>Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), however, one (B-i) and three (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B–C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Diving CM nuclei undergoing chromosomal missegregation in the hypertrophied RVs, with one nucleus receiving three sister chromatids while the other receiving only one chromatid.</p

Endogenous knock-down of RFC40 in rat neonatal cardiac myocytes results in chromosomal missegregation/aneuploidy.

<p> Rat Neonatal cardiac myocytes (RNCMs) were isolated as described previously and grown in 12 well plates and 2-chambered slides for 48 hr. RNCMs were then treated with non-targeting-siRNA (NT) and On-Target plus smartpool RFC40-siRNA respectively, for 72 hr. (<b>A–D</b> and <b>F–G</b>) RNCMs grown in 2-chambered slides were subjected to FISH analysis following treatment with RFC40-siRNA. Untransfected (UT) and RFC40-siRNA-RNCM slides were co-hybridization with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue). RFC40 knock-down was confirmed by performing immunohistochemical analyses for RFC40 (A–D-ii and F–G-ii; Green) in each sample. Merge images are shown in A–D-iii and F–G-iii. Panels A–D represents RNCM nuclei aneuploidy and panels F–G represent chromosomal missegregation. White arrows point to Cen12-ROX signals. Yellow arrow points to micronuclei in G-i & iii. (E) Graph represents the number of signals for Cen12-ROX observed per RNCM nuclei in the UT and RFC40-siRNA treated samples. Fifty RNCM nuclei in control and RFC40-siRNA treated samples from three individual experiments were measured. (H) Before lysing the cells grown in 12-well plates for western blot analyses (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039009#pone.0039009.s004" target="_blank">Fig. S4</a>), the RNCMs (n = 3) were trypsinized, resuspended in 1X PBS and counted using a hemocytometer. Graph represents the number of RNCMs vs the different RNCM treated samples. Values are mean ± SE. * indicates P<0.05 vs. Untransfected (UT).</p

DNA replication proteins are re-expressed in hypertrophied RV.

<p>Total protein lysates (50 µg) obtained from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) were analyzed by 12% or 9% SDS-polyacrylamide gels. (<b>A</b>) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (<b>B and C</b>) Graphs represent summary data for the protein expression of RFC40 and p125 proteins in LV (B) and RV (C) normalized by GAPDH (n = 5). Values are mean ± SE. *indicates P<0.05 vs. control.[/LOOSEST]</p

Up-regulation/re-expression of replication proteins in hypertrophied hearts occurs at the transcription level.

<p>Total RNA (50 ng) extracted from LV and RV tissues (n = 5) of the control (Con), SUHx-3 wks (3 wks) and SUHxNx-5 wks (5 wks) was subjected to real-time one-step-RT-PCR. The amplified products of RFC40, p125, and GAPDH mRNA/cDNA were visualized on 3% agarose gels at the end of each run. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). Graphs represent the changes in the mRNA levels for RFC40-mRNA (A) and p125-mRNA (B) calculated from the crossing point deviation of all the samples and normalized by GAPDH values. Values are mean ± SE. *indicates P<0.05 vs. control.</p

Expression of DNA replication proteins in control and hypertrophied hearts.

<p>All values are mean ±SD; ns-non-significant;</p>*<p>P<0.05 vs control.</p

Cardiac myocyte polyploidy was observed in hypertrophied RVs.

<p>Control (Con) and SuHxNx-5 wks (5 wks) RV sections were used to perform FISH analyses by co-hybridization of the tissues with the Cen12-ROX probe (Red). Nuclei were counterstained with DAPI antifade (Blue) and slides were imaged with Spectral Imaging Software using an Olympus BX61 microscope with 1000X magnification. Two FISH signals for Cen12-ROX was observed in the CM nuclei from the control RV (A-i), whereas two (B-i) and four (C-i) signals were observed in the CM nuclei from the hypertrophied RVs. Localization of the CM nuclei in the control (A-ii) and hypertrophied (B-C-ii) RVs were confirmed by performing immunohistochemical analyses for cardiac Troponin I (Green). (D) Graph represents the number of FISH signals for Cen12-ROX observed per CM nuclei in the control and hypertrophied RVs. Fifty CM nuclei were measured from three individual animals in control and hypertrophied RVs. (E) Graph represents the nuclear area of >100 CM nuclei measured from three individual animals in control and hypertrophied RVs. Values are mean ± SE. *indicates P<0.05 vs. control. (F) In vitro DNA synthesis was carried out using primed M13mp18 ss DNA (100 ng) and protein lysates (20 µg) obtained from fetal, control-RV (Con), SUHx-3 wks-RV and SUHxNx-5 wks-RV tissues, respectively, before (F-i) and after (F-ii) immunodepletion of RFC40. The primer extension products were separated on a 7 M urea-6% (v/v) polyacrylamide gel and visualized by the chemiluminescent nucleic acid detection kit. Lane 1: Biotinylated 2-log DNA ladder (0.5 µg; M) in kilobases (B-lane 1; exposed for only 5 secs to visualize all the bands, is separated by a line). Lane 2: unannealed biotinylated M13 80-mer primer (BLM13P; 1 fmole). Blot is a representative of three such individual experiments.</p

Differential expression of the DNA replication proteins in CMs and CFs.

<p>(<b>A</b>) CMs (n = 5) and CFs (n = 5) were isolated from normal adult heart and lysates (50 µg) were then analyzed by 12% or 9% SDS-polyacrylamide gels. Purity of the two fractions were confirmed by Western blot analyses for Troponin I-C (CMs) and S100A4 (CFs) proteins respectively. (<b>B</b>) Expression of RFC37, RFC38, RFC40, RFC140, PCNA and p125 proteins in the CMs and CFs was determined by Western blot analyses. Whole hearts isolated from 15 day-old fetus (n = 5) were used as a positive control (Ft). GAPDH was used as the loading control. (<b>C</b>) Graphs represent protein expression of RFC40 and p125, normalized by GAPDH in the CMs and fibroblasts, respectively. (<b>D</b>) Total RNA (50 ng) extracted from CMs and fibroblasts pellets (n = 5) was subjected to real-time one-step-RT-PCR. At the end of each run the amplified products of RFC40, p125 and GAPDH mRNA/cDNA were visualized on 3% agarose gels. Total RNA extracted from whole hearts of 15 day-old fetus (n = 5) were used as a positive control (Ft). (<b>E</b>) Graphs represent the mRNA levels in the CMs and CFs calculated from the crossing point deviation and normalized by GAPDH values. Values are mean ± SE. BDL: Below detectable levels.</p