Fig. 4
VTRNA1.1 methylation and processing are altered during cell differentiation. a Treatment regime of keratinocytes using calcium switch assay. b, c qRT-PCR to measure RNA levels of up-regulated (b) and down-regulated (c) markers at 2 and 6 days after calcium treatment compared to the 0 day control. Error bars indicate s.d. (n = 3 qRT-PCRs) ****p < 0.0001, ***p < 0.001, **p < 0.01 multiple t-tests. d Methylation levels (n = 5 BS conversion reactions) at cytosines in RNA isolated from undifferentiated (undiff) and differentiated (diff) primary HK shown as box plots showing all points with minimum to maximum values. ****p < 0.0001 Mann Whitney test. e Log2 coverage (n = 5 BS conversion reactions) of sites in RNA isolated from undifferentiated (undiff) and differentiated (diff) primary HK. f Correlation between methylation levels at cytosines in undifferentiated and differentiated primary HK. Elevated methylation levels at tRNAs (examples in blue) and VTRNA1.1 (red). g, h Methylation levels in VTRNA1.1 (g) and tRNA Leu CAA (h) in the indicated cells (left hand panels) and heat maps (right hand panels) showing methylation levels in the individual replicates. i Light microscope image comparing the morphology of primary HK transfected with a control siRNA (Ctr) or svRNA4 after 4 days of differentiation in high CaCl2. Scale bar: 50 μm. j Western blot detecting KRT10 and OVOL1 in HK transfected with Ctr siRNA or svRNA4 four days after calcium-induction. Tubulin served as loading control. k, l Treatment regimes and transfection (upper panels) of svRNA4 (k) or anti-svRNA4 (l) and qRT-PCR (lower panels) to measure RNA levels of the indicated markers 4 days after calcium treatment. Error bars indicate s.d. (n = 3 qRT-PCRs). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 student’s t-test. Source data are provided as a Source Data file
