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. 2009 Sep;11(9):921–933. doi: 10.1593/neo.09636

Figure 4.

Figure 4

Attenuation of VEGF-C expression by cis-natural antisense RNA of LEDGF/p75 and by LEDGF/p75 siRNA. (A) Illustration of the genomic structure of LEDGF variants. Black boxes indicate exons, and the connecting line indicates introns. The representative sequences shown are AF339083 (LEDGF/p52) and NM_133948.4 (LEDGF/p75). (B) Diagram of the mouse cis-NATs. Accession numbers are as follows (in order of start position from left to right): AK140469, AK038357, AK020824, AK171985, AK053153, AK042735, and AK143096. (C) Expansion of the genomic region covered by the cis-NAT AK042735. The 214-bp-long exon probe that was designed against the cis-NAT of LEDGF is indicated by an empty box. Accession numbers of equivalent transcripts in other species: rat - CB576984; human - AV716383, DA171947; cow - CK778664, BF654277. (D) RT-PCR analysis of RNA from mouse, rat, and human origins performed with LEDGF/p75 antisense-specific primers designed to encompass the exon probe (indicated by an open box 4 in panels A and C and an arrow in panel A). Row antisense/RNA indicates that RT reaction was carried out with no enzyme supplementation and serves as a control to monitor genomic contamination in the samples. (E) Analysis of VEGF-C and LEDGF sense and antisense transcripts by specific RT-PCR in control A549 (-) or stably overexpressing LEDGF antisense construct (+). For amplification of antisense transcripts, sense-specific primers were added to reverse transcription, whereas for the detection of the sense transcripts, antisense primers were added to reverse transcription. Row antisense/RNA is as in panel D. (F) Relative intensity of the bands was normalized against GAPDH and is summarized in the bar graph. (G) Immunoblot assay of VEGF-C, LEDGF/p75, and β-tubulin protein extracted from A549 cells stably transfected with an empty vector (-) or with a construct expressing LEDGF antisense (+). (H and I) H1299 cells were cotransfected with VEGF-C-luciferase reporter and empty vector (pIRES) or vector expressing LEDGF antisense (pIRES-LEDGFas) that reduces the expression of LEDGF/p75. Cells were then incubated with 0.2 mM H2O2 for 12 hours (H) or heated to 42°C for 6 hours followed by an additional 6-hour incubation at 37°C (I; mean ± SD, n = 3). (J) Attenuation of pVEGF-Cwt-Luc reporter activity as a result of knockdown of LEDGF/p75 expression using a specific synthetic siRNA. As a control, a nontarget siRNA was applied (mean ± SD, n = 3).