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Fig. 6 | Molecular Cancer

Fig. 6

From: Convergence of eicosanoid and integrin biology: 12-lipoxygenase seeks a partner

Fig. 6

12-LOX knockdown inhibits β4-mediated 12(S)-HETE production, downstream ERK activation, and cellular invasion. (a-c) Validation of 12-LOX KD in A431 cells. (a) 12-LOX mRNA levels measured by RT-PCR in A431 parental, ns (non-silencing) shRNA control cells, and 12-LOX KD cell lines. *p < 0.001 (b) Western blot analysis of 12-LOX protein levels in 12-LOX KD clones, ns shRNA control, parental A431, CHO (negative control for 12-LOX expression; polyclonal platelet-type 12-LOX antibody appears to be recognizing another 12-LOX isoform in CHO cells), PC-3 12-LOX overexpressors along (positive control for 12-LOX), 3.1 empty vector control cells, and platelet lysate (positive control for 12-LOX expression). (c) No increase in 12(S)-HETE levels were seen with 3E1 stimulation in #1 and #2 12-LOX KD clones. 12-LOX activity was measured by 12(S)-HETE production using LC-MS after a 6 h incubation with 3E1 and AA. (d) #1 12-LOX KD cells do not respond to 3E1 stimulation with an increase in phosphorylated ERK levels. Western blot evaluation of phosphorylated ERK with 30 min 3E1 stimulation. Densitometry analysis represents the ratio of phosphorylated ERK to total ERK. (e) #1 12-LOX KD cell invasion is not affected by BMD122 enzymatic inhibition of 12-LOX. Cells were pre-treated with BMD122, then stimulated with 3E1 or EGF and allowed to invade through a Boyden Chamber insert coated with Matrigel for 24 h. Images taken at 10 x. Invaded cells were stained with crystal violet, the dye content dissolved in 10 % acetic acid, and the absorbance measured at OD570nm. Columns represent the invasion reported as the mean of three samples +/− SE

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