High-content γ/β globin analysis as readout of siRNA screening in β-K562 confirms HDAC as targets for γ-globin activation.

<p>A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HDAC3. Two siRNAs were tested, with two technical replicates. C) β-K562 treated with two different HDAC inhibitors: entinostat and dacinostat (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.s003" target="_blank">S3 Fig</a>). A and C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.g001" target="_blank">Fig 1</a>.</p

HMOX2 siRNA-mediated knockdown and hemin or Tin-PPIX treatment have similar effects on β-K562 hemoglobinization levels.

<p>A) Cells were transfected with a non-targeting oligo (siNTO) as negative control and with a siRNA directed to HMOX2. Two siRNAs (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.s004" target="_blank">S4A Fig</a>) were tested, with two technical replicates. C) Cells were treated with 50μM of either hemin or Tin-PPIX. A, C) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B and D) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.g001" target="_blank">Fig 1</a>. E) RTqPCR on α-, γ- and β-globins from cells treated with hemin or Tin-PPIX. Histograms show levels of globins expression relative to GAPDH (n = 3).</p

High-content analysis of compound-induced changes in globins accumulation.

<p>β-K562 cells were treated with 800μM hydroxyurea and 900μM butyric acid (n = 3, a representative experiment is shown here) and the same cells were analyzed in parallel by immunofluorescence and by RTqPCR 4 days after the addition of the drugs. A) Immunofluorescence images (Bar = 50μm) and relative scatter plots. Data from three independent experiments are presented and statistically analyzed (B) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.g001" target="_blank">Fig 1</a>. C) RTqPCR on α-, γ- and β- globins. Histograms show the relative levels of expression relative to GAPDH. D) Confocal analysis of β-K562 cells untreated or treated with HU as in panel A and subjected to a quadruple staining with Hoechst (blue), anti β- (green), anti γ-globin (red) and anti-CD235a (white). Magnification: 20x. Right panel: 40x magnification of individual cells γ⁺CD235a⁺ or β⁺CD235a⁺ double positive and γ⁺β⁺CD235a⁺ triple positive, respectively.</p

Analysis of γ/β globin levels by immunofluorescence and automated image capture.

<p>A) Image acquisition and analysis for β-K562 and K562. Merged signals of DNA (Hoechst-33342), β-globin and γ-globin are read in channel 1 (Ch1), channel 2 (Ch2) and channel 3 (Ch3), respectively (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141083#pone.0141083.s001" target="_blank">S1D Fig</a>). Bar = 50μm. The intensity value of signals is automatically assigned by the instrument and converted into a corresponding intensity of color. The relative scatter plots show the distribution of double γ^-β^- negative, single γ⁺β^- positive, single γ^-β⁺ positive and double γ⁺β⁺ positive cells (x axis: FITC-β-globin; y axis: PE-γ-globin). Numbers within plots refer to the averaged percentage of cells within each population from three independent experiments (n = 3). The relative st.errors are shown in panel C: *p<0,05; ** p<0,01; ***p<0,001. B) Quantitative fluorescence imaging of single cells: cells numbered from 1 to 6 in panel A are taken as an example of γ^-β^- double negative (1 and 2), single γ⁺β^- positive (5), single γ^-β⁺ positive (4) and γ⁺β⁺ double positive (3 and 6). C) Statistical analysis (n = 3): γ^-β^- cells; red: γ⁺β^- cells; yellow: γ⁺β⁺ cells; green: γ^-β⁺ cells. D) RTqPCR on α, ε, γ- and β-globins. Histograms show the relative levels of expression normalized on glyceraldehyde-3-phosphate dehydrogenase (GAPDH). n≥3, statistical analysis: *p<0,05; **p<0,01; ***p<0,001.</p

Discovery of 2‑(Cyclohexylmethylamino)pyrimidines as a New Class of Reversible Valosine Containing Protein Inhibitors

Valosine-containing protein (VCP), also known as p97 or cdc48 in yeast, is a highly abundant protein belonging to the AAA ATPase family involved in a number of essential cellular functions, including ubiquitin–proteasome mediated protein degradation, Golgi reassembly, transcription activation, and cell cycle control. Altered expression of VCP has been detected in many cancer types sometimes associated with poor prognosis. Furthermore, VCP mutations are causative of some neurodegenerative disorders. In this paper we report the discovery, synthesis, and structure–activity relationships of substituted 2-aminopyrimidines, representing a new class of reversible VCP inhibitors. This class of compounds, identified in a HTS campaign against recombinant VCP, has been progressively expanded and manipulated to increase biochemical potency and gain cellular activity