Journal: Cell Death and Differentiation

Article Title: Connexin43 dephosphorylation at serine 282 is associated with connexin43-mediated cardiomyocyte apoptosis

doi: 10.1038/s41418-019-0277-x

Figure Lengend Snippet: Cx43 expression and distribution in NRVMs transfected with different mutants. a–d NRVMs were transfected with virus carrying Cx43-wt, S279A or S282A gene (20 m.o.i. for each) for 24 h and lysed by RIPA (a, b) or RIPA + junctional/non-junctional isolation treatment (c, d). The lysates were analyzed by Western blot (a, c), and the fold changes in phosphorylated S282 (pS282), pS262, pS368 and Cx43 abundances in relative to those of vector cells (after normalized with GAPDH or β-actin) were thereby detected (b, d). Junctional and non-junctional fractions were referred to cell-membrane and cytosolic fractions to detect Cx43 abundance distributed in both regions (see Methods), respectively, and no difference in their pattern between the two regions was found among the different treatments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired two-tailed Student’s t test, n = 4–5 independent experiments for each group as indicated

Article Snippet: Antibodies used were as follows: rabbit anti-Fas, anti-N-Cad and anti-cytochrome C (Abcam), rabbit anti-pS282-Cx43 and anti-pS279-Cx43 (Biobyt), rabbit anti-Cox IV, anti-p-p38 MAPK (Thr180/Tyr182), anti-p38 MAPKα, anti-pS368-Cx43, anti-HA, anti-pS262-Cx43 and anti-Cx43 (Santa Cruz), and rabbit anti-ZO-1 (Proteintech), mouse anti-GAPDH and anti-β-actin (ZSGB-BIO), mouse anti-TNFR1, anti-caspase-8 and anti-FADD (Santa Cruz), and goat anti-Cx43 (Acris) and anti-DR5 (Santa Cruz).

Techniques: Expressing, Transfection, Isolation, Western Blot, Plasmid Preparation, Two Tailed Test

Journal:

Article Title: Nuclear Heat Shock Response and Novel Nuclear Domain 10 Reorganization in Respiratory Syncytial Virus-Infected A549 Cells Identified by High-Resolution Two-Dimensional Gel Electrophoresis

doi: 10.1128/JVI.78.21.11461-11476.2004

Figure Lengend Snippet: Characterization of A549 cell nuclear preparations. (A to D) Microscopic analysis of sucrose step gradient-purified nuclei. Purified nuclei were diluted in PBS, plated on a microscope coverslip, and stained with DAPI (see Materials and Methods). Panels A and C, phase-contrast microscopy; panels B and D, DAPI staining. Panels A and B, low-resolution images. Panels C and D, high-resolution images. In panel C, nucleoli are visible (arrows). (E) Western immunoblot for nuclear and cytoplasmic markers. Duplicate 75-μg samples of nuclear and cytoplasmic extracts were fractionated by one-dimensional SDS-PAGE, transferred to polyvinylidene difluoride, and stained with lamin B and β-tubulin antibodies. The locations of molecular weight markers (10−3) are indicated on the left. The lamin B stain is localized to the nuclear fraction (Nuc), whereas the β-tubulin is localized to the cytoplasmic lysates (Cyto).

Article Snippet: Anti-lamin-B, -PML, and -Sp100 antibodies were affinity-purified rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif.); anti-β-tubulin (Santa Cruz Biotechnology) and -β-actin (Santa Cruz Biotechnology) were mouse monoclonal antibodies.

Techniques: Purification, Microscopy, Staining, Western Blot, SDS Page, Molecular Weight

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A activates the Wnt/β-catenin pathway by stabilizing β-catenin. A-B , MYC, CCND1 and nuclear β-catenin levels in NSCLC cells as indicated were analyzed by western blotting. C , CTNNB1 transcript levels in NSCLC cells as indicated was analyzed by qRT-PCR. ns, not significant. D , Subcellular localization of SETD1A and β-catenin in NSCLC cells was analyzed by confocal laser scanning microscope. The subcellular distribution of SETD1A and β-catenin was quantified by Image J software. E , The interaction between SETD1A and β-catenin in PC9 cells was analyzed by CoIP assay. F - G , β-catenin stability in the SETD1A knockdown and negative control group PC9 cells was analyzed by CHX chase assay. H , Ubiquitination of β-catenin in PC9 cells was analyzed by CoIP assay following SETD1A knockdown. I , Two-step co-immunoprecipitation of the complex containing SETD1A, β-catenin and PKA is shown. HEK293T cells were cotransfected with the Flag-tagged SETD1A plasmid and HA-tagged β-catenin plasmid. The first immunoprecipitation was performed using anti-FLAG M2 beads. The complex was eluted using 3 × Flag peptide followed by the second step of co-immunoprecipitation with anti-HA antibody. Then the protein samples were analyzed by western blotting with anti-Flag, anti-HA and anti- anti-PKAα cat antibody. Data are shown as means ± SD.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Western Blot, Quantitative RT-PCR, Laser-Scanning Microscopy, Software, Co-Immunoprecipitation Assay, Negative Control, Immunoprecipitation, Plasmid Preparation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A binds to β-catenin via its SET domain, A , Schematic representation of SETD1A mutants. B , HEK293T cells were cotransfected with the indicated plasmids of Flag-tagged SETD1A mutants and HA-tagged full length β-catenin. Cell lysates were immunoprecipitated with anti-Flag antibody. C , The total, cytoplasmic and nuclear β-catenin levels were detected by western blot analysis following transfection with the empty vector, wild-type SETD1A plasmid and ΔSET plasmid. D , Sphere formation ability was determined following transfection with the empty vector, wild-type SETD1A plasmid and ΔSET plasmid. Scale bar, 100 μm. E , Cisplatin sensitivity was detected by the CCK-8 assay following transfection with the empty vector, wild type SETD1A plasmid and ΔSET plasmid. Data are shown as means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, CCK-8 Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A activates NEAT1 and EZH2 transcription to activate the Wnt/β-catenin pathway. A-B , The correlation between SETD1A and NEAT1, EZH2 was analyzed using GEPIA online tool. R, Pearson’s correlation coefficient. C , Schematic showing the ChIP-PCR detection site in the NEAT1 and EZH2 promoters. D , The enrichment of SETD1A and H3K4me3 in the NEAT1 and EZH2 promoters in A549 cells was detected by ChIP-PCR assay. E , The relative enrichment of SETD1A and H3K4me3 in the NEAT1 and EZH2 promoters was detected by ChIP-qPCR assay following SETD1A knockdown in A549 cells. F , The promoter activity was analyzed by luciferase activity assay in A549 cells. G - H , NEAT1 and EZH2 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A knockdown. I , EZH2 protein levels in NSCLC cells as indicated were detected by western blotting following SETD1A knockdown. J-K , NEAT1 and EZH2 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A overexpression. L , EZH2 protein levels in NSCLC cells were detected by western blotting following SETD1A overexpression. Data are shown as means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Activity Assay, Luciferase, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: The SETD1A/β-catenin axis promotes NSCLC progression in vivo. A , A photograph of the tumors collected from nude mice after 28 days ( n = 5). B , The tumor volumes were measured at the indicated time points. C , The tumor weights were measured after xenograft resection. D , Immunohistochemical staining of Ki67, EZH2 and β-catenin in the xenograft specimens. Scale bar, 50 μm. E , The transcript levels of NEAT1, EZH2 and CTNNB1 in the xenograft tissues were detected by qRT-PCR. F , Tumor initiation was monitored for 8 weeks and the tumor initiation frequency was calculated using the ELDA. Data are shown as means ± SD. ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: In Vivo, Immunohistochemical staining, Staining, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: SETD1A is a direct target of the Wnt/β-catenin pathway. A , Schematic showing the putative TCF/LEF binding site in the TCF7L2/TCF4 enriched region. Red line, putative TCF/LEF binding site predicted by PROMO online tool; green region, TCF7L2/TCF4 enriched region derived from ENCODE database. B , The enrichment of β-catenin and TCF4 in the SETD1A promoter region was detected by ChIP-PCR assay. C , The enrichment of β-catenin and TCF4 in the SETD1A promoter region was detected by ChIP-qPCR assay. D , The promoter activity of SETD1A gene in NSCLC cells was analyzed by luciferase activity assay. ns, not significant. E , SETD1A transcript levels in NSCLC cell as indicated were detected by qRT-PCR. H, SETD1A protein levels in NSCLC cells as indicated were detected by western blotting. Data are shown as the means ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: Binding Assay, Derivative Assay, Activity Assay, Luciferase, Quantitative RT-PCR, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: An SETD1A/Wnt/β-catenin feedback loop promotes NSCLC development

doi: 10.1186/s13046-021-02119-x

Figure Lengend Snippet: Schematic diagram of the SETD1A/Wnt/β-catenin positive feedback loop in this study. In NSCLC cells, SETD1A activates the Wnt/β-catenin pathway through two mechanisms as follows: i, SETD1A interacts with and stabilizes β-catenin protein; ii, SETD1A promotes the transcription of NEAT1 and EZH2. In turn, the Wnt/β-catenin pathway activates SETD1A transcription, thus forming a positive feedback loop to coordinately promote NSCLC progression

Article Snippet: The antibodies used for CoIP assay were anti-Setd1A antiboty (A300-289A; Bethyl Lab) and anti-β-catenin antibody (51067–2-AP; Proteintech), mouse anti-Flag (F1804; Sigma-Aldrich), anti-HA (51064–2-AP; Proteintech) and normal rabbit IgG (#2729; Cell Signaling Technology).

Techniques: