Figure 4
Human Wnt5a acts as an ER stress initiator in mammalian cells.
(a) HEK293T cells were transfected with xbp1-GFP and then were treated with Thapsigargin (TG, 2 μM) for 12 h at 24h post-transfection for inducing ER stress. The splicing of xbp1 was detected by anti-GFP antibody. (b) HEK293T cells were co-transfected with xbp1-GFP and wnt5a-flag-KDEL. GFP and Flag antibodies were used for detecting the spliced Xbp1 and hWnt5a, respectively. (c) HeLa cells were transfected with xbp1-GFP, or together with hWnt5a-flag-KDEL at a ratio of 1:3. Portions of xbp1-GFP-transfected wells were treated with TG as positive control. The spliced Xbp1 was indicated by GFP signal. The expression of hWnt5a was indicated by Flag staining (red). (d) The cells with visible GFP expression were calculated and quantified. Data represent the Mean ± SEM (t-test, ***P < 0.001, n = 10 random selected fields). (e) HEK293T cells that stably expressed hWnt5a-Flag were transfected with hWls siRNA. The transfected cells were washed twice at 72hr post-transfection for lysis. Lysate was immunobloted with Grp78, Flag and Tubulin antibodies. (f) The knockdown efficiency of hWls siRNAs was detected by real-time PCR. (g) The relative levels of GRP78 as shown in (e) were quantified after normalization against Tubulin. Values represent the Mean ± S.E.M. (student’s t-test, n = 3, *P < 0.05, **P < 0.01).
