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