Fig 1: Rab8b Is a Direct Downstream Target of p53, which Is Activated by CQ(A) CQ induced Rab8b protein and mRNA levels in a p53-dependent manner. Wild-type (p53+/+) or p53−/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr; and either the lysates were examined by western blot analysis with the indicated antibodies (left panel), or mRNA prepared from the cells was examined by real-time qRT-PCR (right panel). *p < 0.001, by Student’s t test.(B) p53 directly bound to its consensus-binding site in the Rab8b promoter. MEF cells were treated with CQ or vehicle (V) and subjected to ChIP analysis with p53 antibody (p53 Ab) or control IgG antibody, and immunoprecipitated DNA fragments were amplified and analyzed on agarose gels (left panel) or by real-time qPCR (right panel) with primers near the p53-binding site in Rab8b promoter. The immunoprecipitated fragments were similarly analyzed with random primers for GAPDH promoter or two different primer sets for the Par-4 gene, which does not contain a p53-binding site. *p < 0.001, by Student’s t test.(C) Par-4 co-localized with Rab8b+ vesicles in CQ-treated cells. MEFs were treated with vehicle (V) or CQ (25 µM) in the absence or presence of BFA (1 µg/mL) for 24 hr and subjected to ICC analysis for Par-4 (red fluorescence) and Rab8b (red fluorescence). Cells were stained with DAPI to reveal their nuclei (blue fluorescence). Co-localization of Par-4 and Rab8b vesicles in the overlay images is indicated by yellow fluorescence. Note the dissociation of Par-4 and Rab8b (loss of yellow fluorescence but retention of red and green fluorescence) in the CQ + BFA panel. Cells showing co-localization of Rab8b and Par-4 were scored, and the data are expressed as percentage of cells showing co-localization (right panel).Error bars indicate mean of at least three independent experiments ± SD. See also Figure S6.
Fig 2: CQ Induced Par-4 Secretion Is Dependent on Rab8b(A) CQ induced Par-4 secretion by a Rab8b-dependent mechanism. Rab8 wild-type (WT), Rab8b−/−, or Rab8a−/− MEFs were treated with CQ (25 µM) or vehicle (V) for 24 hr, and the CM or lysates were examined by western blot analysis with the indicated antibodies.(B) Induction of Par-4 secretion in response to CQ in Rab8b null cells was restored by re-introduction of Rab8b. Rab8b null MEFs were transiently transfected with GFP-mouse Rab8b (GFP-mRab8b) expression construct or GFP-expression construct for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies.(C) Par-4 secretion in response to CQ was inhibited by Rab8b siRNAs. Wild-type MEFs were transfected with siRNA duplexes from two different sources, Dharmacon (D) and Santa Cruz Biotechnology (SC), or with scrambled siRNA duplexes for control, and the transfectants were treated with CQ (25 µM) or vehicle for 24 hr. The CM and lysates from the cells were examined by western blot analysis with the indicated antibodies.(D) Introduction of human Rab8b in Rab8b-knockdown MEFs resulted in restoration of Par-4 secretion in response to CQ. MEFs were transfected with the indicated siRNAs, 24 hr later, they were re-transfected with GFP-human Rab8b (GFP-hRab8b) or GFP expression construct, and the transfectants were treated for 24 hr with CQ (25 µM) or vehicle. Western blot analysis of the CM and lysates was performed by using the indicated antibodies. Knockdown of endogenous Rab8b was confirmed with the Rab8b antibody, and expression of GFP-hRab8b was detected with the GFP antibody.See also Figure S5.
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