Journal: Communications Biology
Article Title: A C21-steroidal derivative suppresses T-cell lymphoma in mice by inhibiting SIRT3 via SAP18-SIN3
Figure Lengend Snippet: A671 binds SAP18 within the SIN3 suppressor complex to transcriptionally repress SIRT3 expression. a , b Efficient depletion of SAP18 in HEL cells using shRNA as determined by western blotting ( a ) and Q-RT-PCR ( b ). Untreated or scrambled transfected vector (Vector) cells used as control. c Depletion of SAP18 in HEL cells (Sh-SAP18) induces SIRT3 transcription. d Marginal effect of Sh-SAP18 on cell proliferation. e Survival rate of Sh-SAP18 and controls cells treated for 24 h with indicated concentrations of A671. f SIN3A transcription factor (TF) binding sites within the Sirtuin gene family promoters. DNA sequencing data derived from SIN3A chromatin immunoprecipitation (ChIP) for two cell types (GM12878, B-lymphocyte lymphoblastoid, and H1-hESC, human embryonic stem cells) obtained through the ENCODE database (for details see supplementary Fig. 8 ). Of the Sirtuin genes, SIN3A observed to bind within the promoters of all but SIRT7 . The greatest binding observed for SIRT3 was ~108% higher than SIRT1 in GM12878 cells and ~40% higher than SIRT2 in the H1-hESCs. g Expression of SAP18 in HEL cells treated with actinomycin D (10 μM) alone or in combination with A671 (0.5 μM) for 12 (top) or 24 h (bottom). GAPDH used as a loading control. h A three-dimensional view of the predicted interaction of A671 with SAP18. i Position of chemical interactions with the indicated amino acids within SAP18. j Predicted affinity properties of A671 to SAP18 and SIRT3. P
Article Snippet: Similarly, SAP18 (pCMV3-SAP18-GFPSpark®; Sino Biological, China) overexpression studies conducted using Lipofectamine 3000 and hygromycin (250 µg/ml) used for cell selection.
Techniques: Expressing, shRNA, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Binding Assay, DNA Sequencing, Derivative Assay, Chromatin Immunoprecipitation