Spatial distribution of PARP and SATB1 in relation to DNA or XmnIsequence.

<p>(A, D) Whole cells stained by dual-color immunofluorescence. (B, E) NM prepared <i>in situ</i> and stained by immuno-FISH. (A, B) Confocal microscope analysis of the localization of PARP (green) and DNA or XmnI sequence (blue). (D, E) Localization of SATB1 (green) and DNA or XmnI sequence (blue). In the bottom of panels B and E the intensity profile line scans, performed between the white crosses of the NM as indicated on confocal merge images in B and E, are shown. The ordinate represents the fluorescence intensity in arbitrary units, the abscissa represents the distance in pixels. The bars correspond to 5 µm. (C, F) Scatter plots showing quantification analyses of the colocalization of PARP/DNA (a), PARP/XmnI (b), SATB1/DNA (a), or SATB1/XmnI (b), respectively. R corresponds to Pearson’s correlation coefficient; M1 to the fraction of protein being studied overlapping the DNA or XmnI and M2 the fraction of DNA or XmnI overlapping the protein. Horizontal lines show the mean values±SE of 20 fields (122–226 total NMs) replicated in two different experiments (*P≤0.03, **P<0.001).</p

DNA loop organization in LNCaP and PC3 cells.

<p>(A) Representative nucleoids stained either by DAPI (blue) to visualize only total DNA (left panels) or by halo-FISH to highlight the XmnI sequence (red) and counterstained with DAPI to detect total DNA (blue).The bar corresponds to 10 µm. (B) Scatter plot showing the distribution of DNA halo size. Horizontal lines indicate the mean values obtained measuring for each cell line at least 100 nucleoids. The average DNA halo size ± SE was 6.8±0.2 for LNCaP cells and 7.5±0.2 for PC3 cells, respectively (P = 0.009). The bottom panel shows the frequency distribution of the halo radii grouped in intervals of 2 µm. (C) A schematic model of the interrelationship between the loops and the NM in the dedifferentiation of PCa cells. In more-differentiated cells (LNCaP) the NM is well organized with several proteins bound to MAR sequences. In PC3, where some structural regularities of the NM disappear, a smaller number of protein species bound the MARs and so a larger DNA loop is anchored to the NM.</p

NM proteins binding the XmnI sequence in PCa cell lines.

<p>(A) Representative Deep Purple-stained 1D gel and the corresponding SWB. The arrows on the right indicate the three principal bands arising in 1D SWB, each of which corresponds to several spots in 2D as evident in (D), (F) and (H). (B) The comparison between the relative quantity of XmnI binding to NM proteins in the different cell lines. Ordinate represents the mean±SE of the relative amounts of XmnI as determined by quantitative analysis of three different preparations. The decrease in 22Rv1 and PC3 with respect LNCaP cells was significant (*P = 0.004, **P<10⁻⁵). (C, E and G) Representative 2D silver-stained gel maps and (D, F and H) SWB of NM proteins extracted from LNCaP (C, D), 22Rv1 (E, F) and PC3 (G, H) cells. The proteins identified are highlighted in red boxes. The three arrows show the three groups of proteins pointed out in (A). L, lamin; h, hnRNP, fr, fragments.</p

Spatial distribution and phosphorylation level of lamin B in the NM.

<p>(A) Representative confocal microscope images of lamin B (red) and XmnI sequence (blue) in the NM extracted <i>in situ</i> and stained by immuno-FISH. The bars correspond to 5 µm. (B) Magnified section of 2D-PAGE stained with SYPRO Ruby (a, c) or Pro-Q Diamond that selectively stains only phosphoproteins (b, d). The arrowheads indicate the various isoforms of lamin B. The same color corresponds to the same isoform in two cell lines. In PC3 cells, the non-phosphorylated peptide present in LNCaP cells disappeared (red arrowheads).</p

Expression levels of hnRNP U, Matrin3, PARP and SATB1 in LNCaP and PC3 cells.

<p>The ordinates represent the mean±SE of the relative amounts of these proteins as determined by quantitative analysis of three to six WBs carried out utilizing at least three different preparation of NM (*P≤0.05; **P<0.0005). Representative WBs are shown on the right; the major proteolytic fragments of PARP1 and SATB1 are marked by full dots. The relative molecular weights of standard proteins in kDa are reported.</p

Analysis of the differentially expressed NM proteins in LNCaP, 22Rv1 and PC3 cells.

<p>(A) Venn diagram showing the number of protein spots visualized in each cell line. Numbers in the overlapping regions represent common spots. (B–D) Pie charts showing the percentage of spots that bind the XmnI sequence in three cell lines. The colors cyan and yellow, denote the spots that were, with respect LNCaP cells, differently expressed or with no difference, in 22Rv1 (C) or PC3 (D), respectively.</p

Specificity of the antibodies used in this study for WB and SWB analysis.

<p>Specificity of the antibodies used in this study for WB and SWB analysis.</p

AR and hnRNP K are differently distributed in the NM after anti-androgen treatments.

<p>(A) Confocal microscopy analysis clearly shows that in the absence (−) of DHT, the AR is not localized in the NM, whereas in the presence (+) of 0.1 nM DHT, the AR is distributed both in the periphery and in inner punctuate sites. HnRNP K staining is independent of DHT and is present in the internal NM. After exposure of cells grown in presence of 0.1 nM DHT to 10⁻⁵M BIC, both AR and hnRNP K show a very weak diffuse fluorescence pattern. Treatment with 10⁻⁶M CPA gives rise to a strong increase in AR fluorescence. NMs were immunostained with anti-lamin B (blue), anti-AR (red) and anti–hnRNP K (green) antibodies. (B) Scatterplots show the quantification analysis of AR/hnRNP K co-localization. M1 corresponds to the fraction of AR overlapping hnRNP K, and M2 indicates the fraction of hnRNP K overlapping AR; R is Pearson’s coefficient. The horizontal lines indicate the mean values from 20–23 fields (160–190 total cells) replicated in two different experiments; *P<0.02. (C) Frequency distribution of the size of the punctuate sites, corresponding to AR (upper panel) and hnRNP K (lower panel), grouped in intervals of 0.003 µm³. The ordinates are the mean ± SE. The red curves representing BIC treatment are significantly different from control (+DHT; green curves P = 0.01). The blue curves correspond to CPA treatment. Representative projections of image stacks utilized to calculate the size of punctuate sites are reported. hK, hnRNP K. The bars correspond to 10 µm in (A) and 5 µm in (C).</p

Androgen Receptor Activity Is Affected by Both Nuclear Matrix Localization and the Phosphorylation Status of the Heterogeneous Nuclear Ribonucleoprotein K in Anti-Androgen-Treated LNCaP Cells

<div><p>The androgen receptor (AR) plays a central role in the development and progression of prostate cancer (PCa) and anti-androgen therapy is a standard treatment. Unfortunately, after a few years, the majority of patients progress, developing androgen-independent PCa. AR-driven gene transcription recruits a large number of co-activator/co-repressor complexes; among these, the heterogeneous nuclear ribonucleoprotein K (hnRNP K) directly interacts with and regulates the AR translational apparatus. Here we examined AR and hnRNP K expression in response to the treatment of LNCaP cells with anti-androgen cyproterone acetate (CPA) or bicalutamide (BIC). AR and hnRNP K modulation and compartmentalization were studied by Western blot and confocal microscopy. Phosphate-affinity gel electrophoresis was employed to examine how anti-androgens modified hnRNP K phosphorylation. 10⁻⁶M CPA significantly stimulated LNCaP proliferation, whereas for 10⁻⁴M CPA or 10⁻⁵M BIC an antagonistic effect was observed. After anti-androgen treatment, AR expression was remarkably down-regulated within both the cytoplasm and the nucleus; however, when CPA had an agonist activity, the AR associated with the nuclear matrix (NM) increased approximately 2.5 times. This increase was synchronous with a higher PSA expression, indicating that the NM-associated AR represents the active complex. After BIC treatment, hnRNP K expression was significantly lower in the NM, the protein was hypophosphorylated and the co-localization of AR and hnRNP K decreased. In contrast, CPA as an agonist caused hnRNP K hyperphosphorylation and an increase in the co-localization of two proteins. These findings demonstrate that, in vitro, there is a strong relationship between NM-associated AR and both cell viability and PSA levels, indicating that AR transcriptional activity is critically dependent on its subnuclear localization. Moreover, the agonistic/antagonistic activity of anti-androgens is associated with modifications in hnRNP K phosphorylation, indicating an involvement of this protein in the AR transcriptional activity and likely in the onset of the androgen-independent phenotype.</p></div

The PI3K/Akt inhibitor Wortmannin modifies hnRNP K phosphorylation status.

<p>LNCaP cells grown in presence of 0.1(A–D) control (E–H) Wortmannin treated cells. Magnified sections of 2D-PAGE stained with SYPRO Ruby (A, E) or Pro-Q Diamond, that selectively stains only phosphoproteins (B, F), are shown. The boxes mark the phosphorylated hnRNP K isoforms. After Wortmannin treatment a marked decrease in some hnRNP K isoforms and the loss of the more acidic spots are evident. The hnRNP K spots separated by 2D-PAGE (C, G) or 2D-phosphate-affinity-PAGE (D, H) and probed with anti-hnRNP K antibody are also reported. The arrowheads in C, G and the numbers in D, H indicate the different protein isoforms as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079212#pone-0079212-g004" target="_blank">Figure 4</a>. hK, hnRNP K.</p