Journal: Experimental & Molecular Medicine

Article Title: Modulating cellular balance of Rps3 mono-ubiquitination by both Hel2 E3 ligase and Ubp3 deubiquitinase regulates protein quality control

doi: 10.1038/emm.2017.128

Figure Lengend Snippet: Ubiquitinated Rps3p is regulated byUbc4p, Hel2p and Ubp3p in vitro. ( a ) An in vitro ubiquitination assay was performed on purified His 6 -tagged E1 (human), Hel2p, Rps3p, Ubp3p, and ubiquitin. The indicated E2 conjugation enzyme was added into the mixtures. The mixtures were then analyzed by immunoblotting with anti-Rps3p and anti-ubiquitinated Rps3p antibodies. ( b ) The wild-type (wt) and ubc4 Δ strains were grown to the early log phase in YPD media. The cells were exposed to UV stress (+) or not (−) and incubated for 1 h. The whole cell lysates were analyzed by immunoblotting with anti-Rps3p, anti-ubiquitinated Rps3p, anti-eIF2α~p, and anti-Pgk1p antibodies. ( c ) An in vitro ubiquitination assay was performed on purified His 6 -tagged E1 (human), UBCH5b, Hel2p, Rps3p, Ubp3p, and ubiquitin. The Hel2p was added (+) or not (−) into a solution that contained E1, UBCH5b, ubiquitin, and Rps3p (lanes 1 and 2). Ubp3p was added (+) or not (−) into a solution that contained E1, UBCH5b, ubiquitin, Hel2p, and Rps3p. The solutions were analyzed by immunoblotting with anti-Rps3p and anti-ubiquitinated Rps3p antibodies. The Rps3p and ubiquitinated Rps3p are indicated on the right.

Article Snippet: Five hundred nanograms of human ubiquitin-activating enzyme E1 (Enzo), 250 ng of UBCH5b (Enzo), 3 μg of His 6 -Hel2p, 2 μg of His 6 -Rps3p, 3 μg of His 6 -Ubp3p, and 1.5 μg of His 6 -ubiquitin were added to a 50 μl reaction containing 50 m M Tris-HCl (pH 7.5), 150 m M NaCl, 2 m M MgCl 2 , 1 m M dithiothreitol, and 5 m M ATP.

Techniques: In Vitro, Ubiquitin Assay, Purification, Conjugation Assay, Western Blot, Incubation

Journal: Experimental & Molecular Medicine

Article Title: Modulating cellular balance of Rps3 mono-ubiquitination by both Hel2 E3 ligase and Ubp3 deubiquitinase regulates protein quality control

doi: 10.1038/emm.2017.128

Figure Lengend Snippet: Mono-ubiquitination of Rps3p is responsible for cell viability and apoptosis. ( a ) Serial dilutions of the WT or Rps3p K212R mutant cells were spotted onto solid YPD media (lower). Serial dilutions of the WT or Rps3p K212R mutant cells that were exposed to UV stress were spotted onto solid YPD media (upper). The plates were incubated for 2 days at 30 °C ( b and c ). The HT1080 cells stably expressing the Flag-RPS3 wild-type or the K214R mutant were transfected with RPS3 5’-UTR siRNA. After 48 h, these cells were irradiated (or not) with 150 J m −2 UV. ( b ) Subsequently, these cells were labeled with 35 S-Met/ 35 S-Cys (250 μCi per ml) for 15 min. Each cell lysate and ribosomal pellet were then subjected to immunoblot analysis using an anti-ubiquitin antibody (left). To detect the poly-ubiquitinated nascent polypeptides, immunoprecipitation with an anti-ubiquitin antibody was performed for ribosomal pellets, as described in ‘Materials and Methods,’ and subjected to autoradiography (right). ( c ) The UV-irradiated cells were incubated for 8 h, harvested, lysed, and analyzed by immunoblotting with the indicated antibodies. UTR, untranslated region.

Article Snippet: Five hundred nanograms of human ubiquitin-activating enzyme E1 (Enzo), 250 ng of UBCH5b (Enzo), 3 μg of His 6 -Hel2p, 2 μg of His 6 -Rps3p, 3 μg of His 6 -Ubp3p, and 1.5 μg of His 6 -ubiquitin were added to a 50 μl reaction containing 50 m M Tris-HCl (pH 7.5), 150 m M NaCl, 2 m M MgCl 2 , 1 m M dithiothreitol, and 5 m M ATP.

Techniques: Mutagenesis, Incubation, Stable Transfection, Expressing, Transfection, Irradiation, Labeling, Western Blot, Immunoprecipitation, Autoradiography

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Involvement of Oct4 in the pathogenesis of thoracic aortic dissection via inducing the dedifferentiated phenotype of human aortic smooth muscle cells by directly upregulating KLF5.

doi: 10.1016/j.jtcvs.2016.05.036

Figure Lengend Snippet: FIGURE 4. Identification of KLF5 as the target gene of Oct4 in HASMCs with increased expression in aortic tissues from TAD patients. A, qRT-PCR showed that the mRNA levels of KLF5, Nanog, Sox2, Etv5, and BMP4 were significantly upregulated in HASMCs transfected with Ad.Oct4 compared with those transfected with Ad.GFP (n ¼ 3 for each cell group). *P <.05 versus Ad.GFP HASMCs. B, Western blot showed that the protein levels of KLF5 in the Ad.Oct4 group were significantly higher than in the Ad.GFP group (n ¼ 3 for each cell group). C and D, The expressions levels of KLF5 protein were significantly higher in TAD patients (n ¼ 12) than in normal controls (n ¼ 6). *P<.05 versus normal aortas. E, Immunohistochemical staining showed that, compared with normal aorta, the KLF5 expression level increased in the nucleus of HASMCs within the aortic media of TAD patients. Arrows point to positive staining. KLF5, Kr€uppel-like factor 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ad.Oct4, adenovirus overexpressing Oct4; Ad.GFP, adenovirus expressing GFP; TAD, thoracic aortic dissection.

Article Snippet: Immunohistochemistry was performed as described previously.15 The primary antibodies used were anti-Oct4 (1:100, ab19857; Abcam, Cambridge, Mass) and anti-Kr€uppel-like factor 5 (KLF5) (1:50, BA2804-2; Boster, Pleasanton, Calif).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Immunohistochemical staining, Staining, Dissection

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Involvement of Oct4 in the pathogenesis of thoracic aortic dissection via inducing the dedifferentiated phenotype of human aortic smooth muscle cells by directly upregulating KLF5.

doi: 10.1016/j.jtcvs.2016.05.036

Figure Lengend Snippet: FIGURE 5. Oct4 activated KLF5 expression by directly binding to its promoter. A, Schematic of the KLF5 promoter with the Oct4 binding site depicted as triangle. The octamer motif bound by Oct4 is highlighted with red letters. The positions of primer sets used for ChIP assay for the bound site are indicated by short red lines. The plasmid containing 109 to 1017 bp of the KLF5 promoter was constructed. B, After overexpression of Oct4 in HASMCs, ChIP of Oct4 was performed. qRT-PCR was used to determine DNA enrichment in the KLF5 promoter relative to input. Normal rabbit IgG was used as a negative control. All experiments were performed in triplicate. C, The dual luciferase assays showed that Oct4 activated the promoter of pGL3-Pro-WT-KLF5 but had little effect on the activity of pGL3-Pro-MT-KLF5. The results are presented as means standard deviation from 3 independent experiments. **P<.01 versus controls. KLF5, Kr€uppel-like factor 5; Oct4, octamer binding protein-4.

Article Snippet: Immunohistochemistry was performed as described previously.15 The primary antibodies used were anti-Oct4 (1:100, ab19857; Abcam, Cambridge, Mass) and anti-Kr€uppel-like factor 5 (KLF5) (1:50, BA2804-2; Boster, Pleasanton, Calif).

Techniques: Expressing, Binding Assay, Plasmid Preparation, Construct, Over Expression, Quantitative RT-PCR, Negative Control, Luciferase, Activity Assay, Standard Deviation

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Involvement of Oct4 in the pathogenesis of thoracic aortic dissection via inducing the dedifferentiated phenotype of human aortic smooth muscle cells by directly upregulating KLF5.

doi: 10.1016/j.jtcvs.2016.05.036

Figure Lengend Snippet: FIGURE 6. KLF5 mediated the inducement of Oct4 on the phenotypic switching of HASMCs. A, HASMCs were transfected with FAM-siRNA against KLF. The transfection efficiency reached 90%. B and C, qRT-PCR and western blot showed that the expression of KLF5 was significantly inhibited by siKLF5 (n ¼ 3 for each cell group). D and E, qRT-PCR and western blot showed that the mRNA and protein levels of a-SMA and MYH-11 in HASMCs co- transfected with Ad.Oct4 and siKLF5 (Ad.Oct4/siKLF) were significantly higher compared with HASMCs co-transfected with Ad.Oct4 and siScramble (Ad.Oct4/siScramble) (n ¼ 3 for each cell group). *P <.05 versus Ad.Oct4/siScramble. F and G, Wound scratch assay indicated that Ad.Oct4/siKLF5 HASMCs presented significantly lower migration ability. Migrated cells were quantitated. The results are presented as means standard deviation from 3 independent experiments performed in duplicate. *P<.05 versus Ad.Oct4/siScramble. Ad.Oct4, Adenovirus overexpressing Oct4; siKLF5, small inter- fering RNA against KLF5; siScramble, scramble negative control siRNA; a-SMA, a-smooth muscle actin; MYH11, anti-myosin heavy chain 11; KLF5, Kr€uppel-like factor 5.

Article Snippet: Immunohistochemistry was performed as described previously.15 The primary antibodies used were anti-Oct4 (1:100, ab19857; Abcam, Cambridge, Mass) and anti-Kr€uppel-like factor 5 (KLF5) (1:50, BA2804-2; Boster, Pleasanton, Calif).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing, Wound Healing Assay, Migration, Standard Deviation, Negative Control

Journal: The Journal of thoracic and cardiovascular surgery

Article Title: Involvement of Oct4 in the pathogenesis of thoracic aortic dissection via inducing the dedifferentiated phenotype of human aortic smooth muscle cells by directly upregulating KLF5.

doi: 10.1016/j.jtcvs.2016.05.036

Figure Lengend Snippet: FIGURE 7. Expression of Oct4 was significantly higher in TAD patients as compared with normal controls. Overexpression of Oct4 induced a phenotype transition of HASMCs from the contractile type to the synthetic type by directly up-regulating KLF5, expression of which was increased as well in aortic media of TAD patients. Oct4, Octamer binding protein-4; TAD, thoracic aortic dissection; HASMCs, human aortic smooth muscle cells; KLF5, Kr€uppel-like factor 5.

Article Snippet: Immunohistochemistry was performed as described previously.15 The primary antibodies used were anti-Oct4 (1:100, ab19857; Abcam, Cambridge, Mass) and anti-Kr€uppel-like factor 5 (KLF5) (1:50, BA2804-2; Boster, Pleasanton, Calif).

Techniques: Expressing, Over Expression, Binding Assay, Dissection