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. Author manuscript; available in PMC: 2020 Mar 30.
Published in final edited form as: Nat Genet. 2019 Sep 30;51(10):1518–1529. doi: 10.1038/s41588-019-0502-z

Figure 2. NPM1 regulates cellular growth and differentiation through 2’-O-Me.

Figure 2.

a, NPM1-depleted K562 cells (shNPM1) and scrambled transduced cells (Ctrl), were plated in MethoCult. Data are presented as mean ± SD of n = 5 independent experiments. Shown are representative pictures. b, Illustration of Hemin treatment and time course. K562 cells that differentiated to erythrocytes were identified via positive Benzidine staining. c, NPM1 depletion (shNPM1) led to increased differentiation following Hemin treatment. Data are presented as mean ± SD of n = 4 independent experiments. d, Relative levels of snoRNA abundance and 2’-O-Me levels in snoRNA-inactivated cells were calculated relative to the levels in Ctrl cells (K562 transduced with an empty CRISPR/Cas9 vector, set as 1, not shown). Data are presented as mean ± SD of n = 3 independent experiments. e, Data are presented as mean ± SD of n = 4 independent experiments. f, SnoRNA-inactivated K562 cells were treated with Hemin and cellular differentiation was determined relative to Ctrl. Data are presented as mean ± SD of n = 4 independent experiments. g, Scheme of rescue experiments performed by overexpression of individual snoRNAs or of FBL in NPM1-depleted K562 cells. h, snoRNA levels and their corresponding 2’-O-Me levels were analyzed in the relevant RFP+ K562/shNPM1 cells. Data are relative to the levels in Ctrl cells (K562/shNPM1 transduced with an empty RFP vector). Data are presented as mean ± SD of n = 3 independent experiments. i, K562/shNPM1 cells, over-expressing specific snoRNAs (x-axis), were cultured in MethoCult and colony numbers were determined. Data are presented as mean ± SD of 4 independent experiments performed. Statistical significance was determined by two-tailed Student’s t test. j, K562/shNPM1 cells, over-expressing specific snoRNAs (x-axis), were treated with hemin and relative number of differentiated cells was determined by positive Benzidine staining. Data are presented as mean ± SD of n = 4 independent experiments. k, K562/shNPM1 cells, over-expressing FBL, were cultured in MethoCult. Data are presented as mean ± SD of n = 4 independent experiments performed. l, K562/shNPM1 cells, over-expressing FBL, were treated with Hemin and cellular differentiation was determined relative to Ctrl cells (K562/RFP). Data are presented as mean ± SD of n = 4 independent experiments performed. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed Student’s t test.