Skip to main content
. 2016 Mar 1;2:15048. doi: 10.1038/celldisc.2015.48

Figure 6.

Figure 6

The function of TRIP6 in promoting the LPA2 receptor-mediated apoptotic resistance is in part mediated through the activated TRAF6-NF-κB signaling. (a) Knockdown of TRAF6 attenuates the LPA2 receptor-mediated protection from adriamycin-induced apoptosis. The LPA1/2 DKO MEFs stably expressing FLAG-LPA2 with scrambled shRNA or TRAF6 shRNA were pretreated with 10 μm LPA for 1 h, followed by addition of 2 μm adriamycin (Adr) for 8 h. Apoptosis was determined by caspase-3/7 activity assay (top panel). Data shown are the mean±s.e.m. of three independent experiments (*P<0.001 versus Adr treatment; **P<0.01 versus LPA2-shScr MEFs treated with Adr and LPA; Student’s t-test). Another set of cells was subjected to cell viability assay (bottom panel). After treatment, cells were incubated with 0.5 μm calcein-AM and 0.1 μg ml−1 propidium iodide at room temperature for 10 min. Images of calcein-positive green fluorescent live cells and propidium iodide-positive dead cells were acquired under fluorescence microscope. Totally 500–1000 cells per sample were counted to determine the percentage of dead cells. Data shown are the mean±s.e.m. of three independent experiments (*P<0.001, **P<0.01 versus LPA2-shScr MEFs treated with Adr; ***P<0.01 versus LPA2-shScr MEFs treated with Adr and LPA; Student’s t-test). (b) Depletion of TRIP6 eliminates LPA-mediated protection from cisplatin-induced apoptosis in ovarian cancer cells. SKOV-3 stable cell lines expressing Cas9 (sgControl) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) were pretreated with 10 μm LPA in 0.1% fatty acid-free BSA-containing medium for 1 h, followed by addition of 50 μm cisplatin for 20 h. Apoptosis was determined by caspase-3/7 activity assay (top panel). Data shown are the mean±s.e.m. of three independent experiments (*P<0.01 versus sgControl cells treated with cisplatin; Student’s t-test). The percentage of dead cells was determined by cell viability assay using calcein/propidium iodide double fluorescence staining as described above (bottom panel). Data shown are the mean±s.e.m. of three independent experiments (*P<0.05 versus sgControl cells treated with cisplatin; Student’s t-test). (c) Adding TRIP6 back to the TRIP6-depleted SKOV-3 cells restores the antiapoptotic function of LPA; however, this effect is attenuated by the E52A mutation. SKOV-3 cells stably expressing Cas9/TRIP6 sgRNA (SKOV-3-sgTRIP6-1) were transiently transfected with an empty vector (mock), FLAG-TRIP6 or FLAG-E52A-TRIP6. Cells were pretreated with 10 μm LPA for 1 h, followed by addition of 50 μm cisplatin for 20 h. Apoptosis was determined by caspase-3/7 activity assay (top panel). Data shown are the mean±s.e.m. of three independent experiments (*P<0.01, **P<0.05 versus cisplatin-treated cells; ***P<0.05 versus FLAG-TRIP6 cells treated with cisplatin and LPA; Student’s t-test). The percentage of dead cells was determined by cell viability assay using calcein/propidium iodide double fluorescence staining as described above (bottom panel). Data shown are the mean±s.e.m. of three independent experiments (*P<0.05 versus FLAG-TRIP6 cells treated with cisplatin; **P<0.05 versus FLAG-TRIP6 cells treated with cisplatin and LPA; Student’s t-test). The right bottom panels are two representative immunoblots showing the expression of FLAG-TRIP6, FLAG-E52A-TRIP6 or GAPDH in the whole-cell lysates. (d) The effect of TRIP6 knockdown on the inhibition of LPA-mediated antiapoptotic function can be rescued by transiently overexpressing TRIP6; however, this effect is attenuated by the E52A mutation. SKOV-3 cells stably expressing TRIP6 shRNA were transiently transfected with an empty vector (mock), FLAG-TRIP6 or FLAG-E52A-TRIP6. Cells were treated with 10 μm LPA, followed by addition of 50 μm cisplatin for 20 h. Apoptosis was determined by caspase-3/7 activity assay. Data shown are the mean±s.e.m. of three independent experiments (*P<0.01, **P<0.05 versus cisplatin-treated cells; ***P<0.05 versus FLAG-TRIP6 cells treated with cisplatin and LPA; Student’s t-test). The bottom two panels are representative immunoblots showing the expression of endogenous TRIP6, FLAG-TRIP6, FLAG-E52A, or GAPDH in the whole-cell lysates.