LUCL is silenced by DNA methylation. (A) Effects of5-aza-2′-deoxycytidine (5-aza-dC) treatment on LUCH andLUCL. Ten-day-old seedlings grown on plates with or without5-aza-2′-dC were imaged for luciferase luminescence using a CCDcamera. Col-0 was included as the negative control. Each blue or whitespot represents a seedling. Under the same imaging conditions,5-aza-dC-treated LUCL and LUCH seedlings had muchhigher levels of luciferase luminescence compared to mock (DMSO)-treatedseedlings. (B) RT-PCR of mock-treated and5-aza-2′-dC-treated LUCL and LUCH seedlings in(A). The LUC and NPTII genes are shown.UBQ5 served as an internal loading control.‘–RT’ indicates RT-PCR conducted in the absence ofreverse transcriptase during the reverse transcription step. (C)Detection of DNA methylation in LUCH and LUCL by McrBCdigestion of genomic DNA followed by PCR. The + gels are DNAtreated with McrBC. The − gels are DNA treated in thesame manner as the + gels except that no McrBC was added.At2g19920 was used as an unmethylated internal control.(D) The d35S::LUC-AP2 transgene in bothLUCH and LUCL. The four lines below the rectanglesmark the four regions interrogated by bisulfite sequencing in(E). (E) Detection of DNA methylation at the luciferasereporter gene in LUCH, LUCL, LUCL ago4-6 andLUCL drm2-6 by bisulfite sequencing. The graphs representthe percentage of DNA methylation (y-axis) at the three differentcytosine contexts (x-axis). The percentage of DNA methylation is alsolisted in the tables below the graphs. See Additional file 1: Table S2 for bisulfite conversion rates.5-aza-dC: 5-aza-2′-deoxycytidine; RT-PCR: reversetranscription-PCR. DMSO: Dimethyl sulfoxide; McrBC PCR: digestion ofgenomic DNA by McrBC followed by PCR.