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. 2016 Mar 1;2:15048. doi: 10.1038/celldisc.2015.48

Figure 1.

Figure 1

Depletion of TRIP6 eliminates LPA-induced NF-κB and MAP kinase activation in ovarian cancer cells. (a) TRIP6 associates with TRAF6 constitutively in SKOV-3 cells. SKOV-3 cells were starved for 24 h, followed by stimulation with LPA for 20 or 40 min. Endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting with anti-TRIP6 rabbit antibody. The blot was reprobed with anti-TRAF6 rabbit antibody. The bottom panel shows the expression of endogenous TRIP6 in the whole-cell lysates. (b) Knockdown of TRIP6 attenuates LPA-induced IKK activation and IκBα phosphorylation. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were starved overnight, followed by stimulation with LPA for 30 or 60 min. Immunoblotting was performed to determine the levels of phospho-S32/36-IκBα, phospho-S176/180-IKKα/β, total IκBα, IKKβ, TRIP6 or TRAF6 in the whole-cell lysates. (c) Knockdown of TRIP6 reduces LPA-induced JNK activation. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were starved overnight, and then treated with LPA for 20, 40 or 80 min. Immunoblotting was performed to detect phosphorylated or total JNK, p38, TRIP6 or TRAF6 in the whole-cell lysates. (d) Depletion of TRIP6 by CRISPR/Cas9/sgRNA-mediated targeting of TRIP6 gene greatly eliminates LPA-stimulated IκBα phosphorylation and MAP kinase activation. SKOV-3 stable cell lines harboring Cas9 alone (sgControl) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) were treated with LPA for 30 or 60 min. Immunoblotting was performed to determine the levels of phosphorylated or total IκBα, IKKα/β, JNK, p38, ERK or TRIP6 in the whole-cell lysates. (e) Depletion of TRIP6 reduces LPA-promoted nuclear translocation of NF-κB p65. SKOV-3 cells harboring Cas9 alone (sgC) or Cas9/TRIP6 sgRNA (sgT-1, sgT-2) were starved overnight, followed by LPA stimulation for 90 min. Subcellular fractionation was performed in hypotonic buffer to separate the nucleus (pellet) from the cytosol (supernatant). The protein levels of NF-κB p65 or TRIP6 in each fraction were determined by immunoblotting. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Histone H3 serve as cytosolic and nuclear markers, respectively. Data shown in (a–e) are representative of two to four independent experiments. (f and g) Depletion of TRIP6 reduces the basal and LPA-promoted NF-κB activity in SKOV-3 cells. SKOV-3 cells stably expressing shRNA (shScr, shTRIP6) (f) or Cas9/sgRNA (sgControl, sgTRIP6-1, sgTRIP6-2) (g) were transfected with the expression vectors of NF-κB-Luc and β-galactosidase. Cells were starved for 24 h, followed by LPA stimulation for 3 h. The NF-κB-driven luciferase activity was measured and normalized to the β-galactosidase activity. Data shown in (f) are the mean±s.e.m. of six independent experiments (*P<0.001 versus untreated shScr cells; **P<0.001 versus treated shScr cells; Student’s t-test). Data shown in (g) are the mean±s.e.m. of four independent experiments (*P<0.001, **P<0.05 versus untreated cells; ***P<0.001 versus treated sgControl cells; #P<0.001 versus untreated sgControl cells; Student’s t-test). (h) Knockdown of TRIP6 attenuates IL-6 activity in SKOV-3 cells. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were transfected with the expression vector of β-galactosidase and either pIL-6-Luc or pIL-6 mut-Luc with mutation in the NF-κB binding site. After stimulation for 3 h, the IL-6-driven luciferase activity was measured and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of three independent experiments carried out in duplicates or triplicates (*P<0.05, **P<0.01 versus untreated shScr cells; ***P<0.01 versus treated shScr cells; Student’s t-test). (i) Depletion of TRIP6 attenuates LPA-induced Bcl-xL expression. SKOV-3 cells stably expressing Cas9 alone (sgControl) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) were starved overnight, followed by LPA stimulation for 4 or 8 h. Immunoblotting was performed to detect the levels of Bcl-xL, TRIP6 or GAPDH. The result shown is representative of two independent experiments.