Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: SGK1-Mediated Vascular Smooth Muscle Cell Phenotypic Transformation Promotes Thoracic Aortic Dissection Progression

doi: 10.1161/ATVBAHA.124.321421

Figure Lengend Snippet: SGK1 (serum- and glucocorticoid-regulated kinase 1) regulates contractile-to-synthetic phenotypic transformation of mVSMCs and interacts with SIRT6 (sirtuin 6). A through G , Control or Sgk1 small interfering RNA (siRNA)–transfected vascular smooth muscle cells (VSMCs) after 48 hours. A , Real-time quantitative polymerase chain reaction (RT-qPCR) data showing the relative mRNA expression levels of the indicated genes in Sgk1 knockdown mVSMCs. The mRNA levels were normalized to those of Gapdh. B , Western blot analysis of the indicated proteins in Sgk1 knockdown mVSMCs. β-Actin served as a loading control for Western blotting. C and D , Representative immunofluorescence staining for the contractile markers Tagln ( C ) and α-Sma ( D ) in Sgk1 knockdown mVSMCs. nDNA was stained with DAPI (4′,6-diamidino-2-phenylindole). Scale bar=50 µm. E , Sgk1 knockdown mVSMCs were incubated with 5-ethynyl-2′-deoxyuridine (EdU) for 3 hours. A fluorescence microscope was used to detect EdU ( left ), and the results were statistically analyzed ( right ). nDNA was stained with DAPI. Scale bar=100 µm. F , Representative images of SA-β-gal (senescence-associated β-galactosidase)–stained Sgk1 knockdown mVSMCs ( left ) and statistical analysis ( right ). The green regions are positively stained. Scale bar=200 µm. G , Western blot analysis of senescence markers in Sgk1 knockdown mVSMCs. β-Actin served as a loading control for Western blotting. H , Immunoaffinity purification and mass spectrometry analysis of SGK1-interacting proteins. Whole-cell extracts from HEK-293T cells stably expressing FLAG (vector) or FLAG-SGK1 were immunopurified using anti-FLAG affinity columns and eluted with the FLAG peptide. The eluates were resolved using SDS-PAGE and silver stained. Protein bands were retrieved and analyzed using mass spectrometry. I , Mass spectrometry analysis of SGK1-interacting proteins. J , Western blot analysis of the purified fractions using antibodies against SIRT6. K , Coimmunoprecipitation (Co-IP) assay of endogenous SGK1 and SIRT6 in HEK-293T, MOVAS, and mVSMC cells. L , Immunoprecipitation (IP) assay in HEK-293T cells ectopically expressing the indicated proteins. M , Normally cultured VSMCs were fixed and analyzed by immunofluorescence using antibodies specific for SGK1 and SIRT6. nDNA was stained with DAPI. Scale bar=100 µm. N , Glutathione S-transferase (GST) pull-down assays with bacterially expressed GST-fused proteins and in vitro transcribed/translated proteins. O , Domain architectures of SIRT6. P , Identification of the essential domains required for interaction. Q , In vitro kinase assay using recombinant human active SGK1 and GST-fused SIRT6 as substrates. R , IP analysis of the serine phosphorylation of SIRT6 in the total lysates of vector- and SGK1-S422D–transfected HEK-293T cells. S , Sequence alignment of the SGK1 phosphorylation motif of SIRT6 from various species. T , IP analysis of HEK-293T cells revealed that SGK1 phosphorylates serine in wild-type (WT) SIRT6 but not in SIRT6-S338A. Data were presented as the mean±SD of 3 independent experiments. Statistical analyses were performed via 2-tailed unpaired t test. α-Sma indicates alpha smooth muscle actin; FLAG, a peptide tag consisting of eight amino acids (DYKDDDDK); MOVAS, mouse aortic vascular smooth muscle cell line; and mVSMC, primary mouse smooth muscle cells.

Article Snippet: HEK-293T (human embryonic kidney 293T) cells and MOVAS (mouse aortic vascular smooth muscle) cell lines were obtained from Procell Life Science & Technology.

Techniques: Transformation Assay, Control, Small Interfering RNA, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Knockdown, Western Blot, Immunofluorescence, Staining, Incubation, Fluorescence, Microscopy, Immunoaffinity Purification, Mass Spectrometry, Stable Transfection, Plasmid Preparation, SDS Page, Purification, Co-Immunoprecipitation Assay, Immunoprecipitation, Cell Culture, In Vitro, Kinase Assay, Recombinant, Sequencing