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. 2013 Aug 9;41(20):9424–9437. doi: 10.1093/nar/gkt717

Figure 3.

Figure 3.

Fusion of the 132-bp trigger to the luciferase reporter gene results in generation of functional luciferase AS sRNAs. The 19-trigger-luc cell line is E. histolytica HM-1:IMSS parasites stably transfected with the plasmid containing the EHI_197520 trigger fused to luciferase. (A) Schematic of constructs used in this assay. (B) High-resolution northern blot of the 19-trigger-luc and untransfected (HM-1:IMSS) cell lines probed for luciferase AS sRNAs (90 µg of RNA enriched for sRNAs). AS sRNAs to the luciferase reporter gene were detected in abundance, which decreased substantially over time with drug removal. An abundant population of AS sRNA to the EHI_197520 trigger was detected, which increased in the 19-trigger-luc cell line. Loading control: sRNAs to EHI_118130. (C) Transient transfection of the 19-trigger-luc cell line with a luciferase reporter construct (CS-luc). The construct expresses well in the untransfected cell line but does not express in the 19-trigger-luc cell line. Ten days after drug release the CS-luc construct can be partially expressed, and 21 days after drug release a full recovery of CS-luc expression is observed. The values were normalized to a CS-Renilla-luc expression. Experiments were done in triplicate; average and standard errors are shown.