Concatemeric Broccoli reduces mRNA stability and induces aggregates
Fig 5
Polyclonal Broccoli-expressing cell lines showed reduced mCherry expression and no detectable Broccoli fluorescence.
(A) Flow cytometry measurement of mCherry intensity in WT, mCherry or 16xBroccoli 293T or HeLa cells. (B) Northern Blot of mCherry RNA in the stable cell lines. RNA intensity was quantified and calculated relative to the 16S ribosomal band. All quantifications were further compared to the mCherry RNA in the mCherry-293T cells which was normalized to one. (C) RT-qPCR of mCherry. RNA was isolated from the stable cell lines, cDNA prepared and mCherry quantified by qPCR. Delta delta Ct of mCherry was calculated using GAPDH as the reference gene. Afterwards, all values were normalized to mCherry 293T cells. (D) Standard PCR for Broccoli from DNA isolated from 293T and HeLa cells expressing mCherry or 16x Broccoli (four lanes on the left side); lentiviral plasmid used for the generation of the mCherry and 16x Broccoli cell lines (two lanes on the right side). Contrast of gel image was optimized using the substract background function in Fiji (Fiji.sc). Statistical analysis performed by t-test (C). * P<0.05, ** P<0.01.