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. 2019 Jun 11;10:2542. doi: 10.1038/s41467-019-10508-2

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — PSPH suppression blocks the expansion potential of human T-ALL cells in vitro. a Fluorescent immunoblot analysis of PSPH protein levels in KE37, DND41, and RPMI8402 cells upon knockdown of PSPH using two independent shRNAs, with the quantification of three independent blots on the right. b Left: growth curves representing proliferation of PSPH knockdown cells over time for T-ALL cell lines KE37, DND41, and RPMI8402. Middle: Proliferation index which was calculated based on pooling of at least three individual data points from the left plot in order to quantify the effects of PSPH shRNA interference on T-ALL cell proliferation. Right: Apoptosis in PSPH knockdown cells (three averaged data points). Data are represented as mean ± standard deviation. c Immunoblot analysis of phosphorylated CDK2 at threonine 160. d Left histograms: BrdU incorporation or PI cell cycle flow cytometry analysis of representative scrambled control and PSPH knockdown T-ALL cell lines. Right: Quantification of the percentage cycling cells in cultures of scrambled, shPSPH#1, and shPSPH#2 KE37, DND41, and RPMI8402 T-ALL cells. For technical reasons, some T-ALL lines were only analyzed by either BrdU or PI cell cycle analysis, and at least in two independent experiments per sample. e Relative formate-derived NAD(P)H levels in scrambled control and PSPH knockdown T-ALL cells (combined results for KE37, RPMI8402, DND41, X12). Background NAD(P)H levels were subtracted from formate-derived NAD(P)H levels and the data were corrected for protein input. The box-plots include combined results of two independent experiments. f Flow cytometry analysis of de novo protein synthesis by O-propargyl-puromycin (OPP) incorporation. Relative protein synthesis as shown in the figure was calculated as shPSPH#1 and shPSPH#2 OPP MFI relative to scrambled control cells for KE37, RPMI8402, and DND41. All box-plots show the median and error bars define data distribution. Statistical analysis *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. p-values were calculated using a two-tailed Student’s t-test