Supplementary Fig. 3: NPM1-bound snoRNAs modulate translation.
From: Germline NPM1 mutations lead to altered rRNA 2′-O-methylation and cause dyskeratosis congenita
a-d, NIH/3T3 cells were stably transfected with vectors of the PiggyBac transposon system (SBI) to express mouse Snord47 and Snord52, a, Relative snoRNA abundance. Data are presented as mean±SD, n=3 independent experiments. b, Fold increase in 2′-O-Me in snoRNAs overexpressing cells. Data are presented as mean±SD, n=3 independent experiments. c, WB of p27 and XIAP in snoRNA overexpressing cells. RPL22 served as a loading control. Blot is a representative of n=3 independent experiments. d, levels of Cdkn1b and **ap in snoRNA overexpressing cells. Data are presented as mean±SD, of n=3 independent experiments. Actb served as a control. e-g, Npm1+/+ MEFs were transfected with specific anti-snoRNAs GapmeRs and anti-GFP as control (50nM, Exiqon). e, SnoRNA levels 48hrs after transfection. Data are presented as mean±SD of n=3 independent experiments. f, Levels of the IRES-translated genes XIAP and p27 were reduced due GapmeRs transfection. Blot is a representative of n=3 independent experiments. RPL22 served as a loading control. g, Cdkn1b and **ap transcripts levels 48 hours after GapmeRs transfection. Data are presented as mean±SD of n=3 independent experiments. h, WB analysis of FLAG expression levels of NPM1, NPMc+ and NPM-RARα in dual luciferase reporter assays. Blot is a representative of n=2 independent experiments. i, Levels of specific 2′-O-Me residues in OCI-AML3 cell line compared to OCI-AML2. Data are presented as mean±SD of n=3 independent experiments. j, SnoRNA abundance in OCI-AML2 (set as 1) and OCI-AML3 cell lines. Data are presented as mean±SD of n=3 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.