Characterization of Sin1 isoforms.

<p>A. Schematic map representing of the human Sin1 gene and transcript variants described in this study. There are five Sin1 transcript variants generating four distinct protein isoforms. Two transcript variants encode for Sin1δ. The green and red solid regions represent start and stop codon respectively. B. Sin1 isoforms are widely express in mouse tissues. Total RNA extracted from mice tissues were analyzed by RT-PCR using either Sin1γ specific primers or primers recognized all four isoforms, 18s RNA were used as loading control. Amplified products were separated by electrophoresis in a 2% agarose gel stained with gel red, demonstrating that all four Sin1 isoforms are widely expressed in mice tissues.</p

Sin1γ colocalizes with γ-tubulin.

<p>A. Sin1-/- MEF cells were transiently transfected with indicated GFP-tagged expression plasmid, the slides were fixed, stained with anti-γ-tubulin/Texas red-labelled anti-mouse secondary antibody and analyzed by confocal microscopy after 24h transfection. B. Hela cells were transiently transfected with indicated GFP-tagged expression plasmid, the slides were fixed, stained with anti-γ-tubulin/Texas red-labelled anti-mouse secondary antibody and analyzed by confocal microscopy after 24h transction. Magnification of colocalization between GFP- Sin1γ and γ-tubulin was shown on the right panel. C. Sin1γ expression fluctuates during cell cycle. HeLa cells transfected with GFP empty vector or GFP-Sin1γ for 24h were subjected to time-lapse microscopy observation. Both GFP and DIC channels were shown for cells transfected with each expression plamids. Scale bars: 25 μm (A and C), 5 μm (B). All experiments were repeated for three times with the same results.</p

Knockdown Sin1 protein inhibits cilia formation.

<p>A. The percentage of RPE1 cells with cilia were determined using acetylated-tubulin staining. Average data obtained from two to three different views are shown. Approximately 300 cells for each siRNA transfection were scored each time. Error bar represent SD. *p<0.01 compared with luciferase, #p<0.05 compared with luciferase. B. siRNA knockdown efficiency from (A) were detected using western blot.</p

Restoration of Sin1-/- MEFs with GFP-Sin1 expression vectors.

<p>A. Sin1 isoforms exert distinct functions in restoration of mTORC2 signaling. Sin1-/- MEF cells transfected with GFP empty vector, GFP-Sin1α, GFP-Sin1β, GFP-Sin1γ, GFP-Sin1δ respectively were grown in starved, or starved then restimulated with insulin or serum for 15min. Total cell lysates were analyzed for indicated proteins by immunoblotting. B. The MAPK pathway is not altered in Sin1 isoform-rescued Sin1-/- MEF cells. Sin1-/- MEF cells transfected with GFP empty vector, GFP-Sin1α, GFP-Sin1β, GFP-Sin1γ, GFP-Sin1δ respectively were grown in starved, or starved then restimulated with insulin or serum for 15min. Total cell lysates were analyzed for indicated proteins by immunoblotting. Single-letter abbreviations for the treatments are as follows: S, starvation for 12hr, I, insulin for 15min after starvation, F, FBS for 15min after starvation. C. The quantifications analysis of the phosphor-PKC band are shown.</p

Sin1 isoforms except Sin1δ form mTORC2 complex.

<p>A. Rictor pulled down Sin1α, Sin1β, Sin1γ, but not Sin1δ. HEK-293T cells were transiently transfected for 24h with HA-Sin1 isoform plasmids. Cell lysates (left-hand side) and rictor immunoprecipitates (right-hand side) were analyzed for mTOR, rictor and HA by western blotting. B. Sin1α, Sin1β, Sin1γ, but not Sin1δ could pull down rictor and mTOR. HEK-293T cells were transiently transfected for 24h with HA-Sin1 isoform plasmid respectively. Cell lysates (left-hand side) and HA immunoprecipitates (right-hand side) were analyzed for mTOR, rictor and HA by western blotting. All experiments were repeated for three times with the same results.</p