Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Differentially expressed genes (DEGs) from the RNA-seq analysis of WT and Sens3-KO livers were presented by the volcano plot (A). A selected list of DEGs were presented in the table (B). Top 10 biological processes were over-represented in the significantly upregulated (C) and downregulated (D) genes. Hepatic Cd44 and Cd133 mRNA analysis by real-time qPCR (E). Immunoblot analysis of cancer signaling-related proteins in the liver of WT and Sesn3 KO mice (F). Co-IP analysis of Sesn3 and Gli2 interaction in HEK 293T cells (G). Data are expressed as mean ± SEM (n=4 for the immunoblot analysis, n=10 for ELISA, n=6 for qPCR analysis, respectively). #p < 0.05 and ##p < 0.01 for KO vs. WT.

Article Snippet: After an overnight incubation, cells were transfected with different combinations of plasmids including vector, mouse Flag-Sesn3, and mouse HA-Gli2 (Addgene #37671, a gift from Dr. Philip Beachy [ 32 ]), in the presence or absence of a smoothened agonist (SAG, 100 nM, Sigma).

Techniques: RNA Sequencing Assay, Western Blot, Co-Immunoprecipitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Biochimica et biophysica acta. Molecular basis of disease

Article Title: Sesn3 deficiency promotes carcinogen-induced hepatocellular carcinoma via regulation of the hedgehog pathway

doi: 10.1016/j.bbadis.2019.07.011

Figure Lengend Snippet: Representative immunofluorescent images of Sesn3 and Gli2 after vector, Flag-Sesn3, and HA-Gli2 plasmid DNAs were transfected to Huh7 cells in the absence or presence of 100 nM SAG. Sesn3 and Gli2 were detected using anti-Sesn3 or anti-HA antibody followed by Alexa 594 and Alexa 488 secondary antibody, respectively (A, B). Vector or Flag-Sesn3 plus HA-Gli2 were transfected to Huh7 cells in the presence of 100 nM SAG. Sesn3, Gli2, and CD44 were detected using anti-Flag, anti-HA, and anti-CD44 antibody followed by Alexa 594, Alex 488, and Cy5 secondary antibody, respectively (C). Fluorescence images were captured using a Zeiss fluorescence microscope at x640 magnification. Real-time PCR analysis of endogenous GLI2 and CD44 mRNAs in vector or Sesn3 transfected Huh7 cells treated with vehicle or SAG (100 nM) for 24 hours (D, E). Data are expressed as mean ± SEM (n = 3). ###p < 0.001 for vehicle vs. SAG for vector or Sesn3 transfection, and ***p < 0.001 for vector vs. Sesn3 transfection after the SAG treatment.

Article Snippet: After an overnight incubation, cells were transfected with different combinations of plasmids including vector, mouse Flag-Sesn3, and mouse HA-Gli2 (Addgene #37671, a gift from Dr. Philip Beachy [ 32 ]), in the presence or absence of a smoothened agonist (SAG, 100 nM, Sigma).

Techniques: Plasmid Preparation, Transfection, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction