Characterization of the slowest-migrating species of AGS3 and its sensitivity to mTOR-dependent autophagy.

<p>DNA transfections were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#pone-0008877-g001" target="_blank">Figure 1</a>. (A) Western blot analysis of AGS3 immunoprecipitated from HeLa cell lysates in the presence or absence of Antarctic phophatase. (B) Western blot analysis of a truncated AGS3 construct (HA-AGS3TPR; a.a. 1–472) consisting of the N-terminal TPR domains immunoprecipitated from HeLa lysates. The lower light band likely represents a non-specific signal as it appears in both lanes. (C) Western blot analysis of the phosphatase treatment of a truncated AGS3 construct (HA-AGS3GPR; a.a. 461–650) consisting of the C-terminal GPR domains immunoprecipitated from HeLa lysates. (D) The influence of mTOR-dependent and –independent autophagic inductions on the triplet of AGS3. Autophagy was induced in HeLa cells by inhibiting mTOR activity, via either rapamycin (100nM, 4 hrs) or starvation (2 hrs), or by an mTOR-independent pathway via LiCl (10 mM, 24 hrs).</p

A rabbit polyclonal antibody detects an AGS3 post-translational modification in HEK293 and HeLa cells.

<p>DNA and siRNA transfections were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#pone-0008877-g001" target="_blank">Figure 1</a>. (A) In HeLa cells, exogenously over-expressed untagged-AGS3 as well as endogenous AGS3 exhibited three differently migrating bands (indicated by asterisks). (B) Characterization of the putative AGS3 bands using lysates collected from HeLa cells transfected with AGS3 siRNAs. The presence of three differently migrating bands which are sensitive to the AGS3 siRNAs is also evident in HEK293 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#pone-0008877-g001" target="_blank">Figures 1C, 1E</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#pone-0008877-g002" target="_blank">2A</a>). (C) Analysis of the band pattern of an HA-tagged AGS3 expressed in HeLa cells. (D) Comparison of the mobility between recombinant AGS3 expressed in <i>E. coli</i> and endogenous AGS3 from HeLa cell lysate.</p

Both knock-down and over-expression of AGS3 lead to an increased LC3II level in HEK293 and HeLa cells.

<p>Cells transfected with DNA (0.4 µg/ml) or siRNA (20 nM) were lysed 24 or 48 hrs after transfection, repectively, unless otherwise noted. β-actin was used as a loading control. (A) The effect of rapamycin (100 nM, 4 hrs) on the level of LC3II in HEK293 cells. (B) The impact of AGS3 over-expression on LC3II in HEK293 cells. Cells transfected with an empty vector (pcDNA3) were used as a negative control. (C) The influence of AGS3 knock-down on LC3II in HEK293 cells. Cells transfected with a non-targeting siRNA were used as a negative control. (D) The influence of AGS3 knock-down on LC3II in HeLa cells. Western blot LC3II band intensities are normalized to that of the control siRNA treated cells (arbitrarily set as 1X). (E) HEK293 cells were treated with either a non-targeting control siRNA or AGS3 siRNA1 and either starved (2 hrs) or not. (F) GFP-LC3 stable HEK293 cells were treated with either a non-targeting control siRNA or AGS3 siRNA1 and endogenous LC3 was assayed by western blot. Western blot band intensities are normalized to that of the control siRNA treated cells (arbitrarily set as 1X). (G) HEK293 cells stably expressing GFP-LC3 were treated with AGS3 siRNA1 (50 nM, 72 hrs) and either starved (2 hrs) or not; a higher AGS3 knock-down efficiency was obtained for the HEK293 GFP-LC3 stable cells when using a higher siRNA concentration and a longer incubation time. This could be due to differences in cell origin. The number of GFP positive puncta structures were quantified as described in the “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#s2" target="_blank">Materials and Methods</a>”. For the sake of clarity normalized GFP-LC3II counts/cell (n = 6, approximately 10,000 cells/condition) are shown. Two statistical tests (ANOVA and single-sample Student's t-test) performed on LOG transformed data revealed that the AGS3 effect was significant across all experimental conditions (p<0.05 for Starved, p<0.01 for all others). The images on the left side are representative full size acquired images, the inset to the right shows a close-up of one cell with indication of detected GPF puncta (red circles). The green dotted lines enclose the cell nuclei. Representative blots and images are shown (AGS3 was detected using a rabbit polyclonal antibody characterized below). Data presented in bar graphs is the average result from at least three independent experiments except in (G). Error bars: standard error of the mean. Asterisks - * = p<0.05, ** = p<0.01, paired Student's t-test.</p

Over-expression of the AGS3 phospho-mutant does not induce autophagy in HEK293 HeLa or COS7 cells.

<p>DNA transfections were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008877#pone-0008877-g001" target="_blank">Figure 1</a>. (A) Western blot analysis of the AGS3 GPR phospho-mutant (all Ser and Thr within last the C-terminal 183 a.a. mutated to Ala) expressed in HEK293 cells. (B) Co-immunoprecipitation of Gαi2/Gαi3 with either wild-type AGS3 or the AGS3 phospho-mutant from HEK293. (C) Assessment of the ability of AGS3 GPR phospho-mutant to induce autophagy in HEK293 cells. HEK293 cells transfected with the vector alone, AGS3 or AGS3 GPR phosphor-mutant were lysed and LC3II levels was assayed. (D) Assessment of the ability of AGS3 GPR phospho-mutant to induce autophagy in COS7 and HeLa cells. Representative blots are shown. Data presented in the bar graph in (B) are the average results from at least three independent experiments. Error bars: standard error of the mean. Asterisks = p<0.01, paired Student's t-test. Western blot band intensities in (D) are shown as normalized to that of vector (i.e. pcDNA3) transfected cells (arbitrarily set as 1X).</p

Influence of USP9x knockdown on the distributions of marker proteins of ER, Golgi and lysosomes.

<p>(A) HEK293 cells infected by the lentivirus expressing pLVX-shRNA1 (non-targeting), USP9x-shRNA2, or USP9x-shRNA3 were co-stained with anti-USP9x (1 µg/ml) and anti-β-GalT1 (1∶2500) (the top panels) or co-stained with anti-β-GalT1 (1∶2500) and anti-TGN46 (1∶2500) (the bottom panels). (B) Percentage of cells with condensed or dispersed TGN46 staining. The values on the bar graph represented the average from three independent experiments with more than 500 cells were counted in each experiment (error bars: standard deviation). (C–E) The same set of HEK293 cells were stained with anti-Calreticulin (1∶1000; C), p115 (1∶600; D) or Lamp1 (1∶3000; E), respectively. Scale bar: 20 µm.</p

Co-regulation of AGS3 and USP9x in the prefrontal cortex (PFC) of rats after prolonged cocaine withdrawal.

<p>Experimental rats (n = 9) received once/daily i.p. injections of 15 mg/kg cocaine for 1 week followed by 3 weeks of withdrawal. Control rats (n = 9) were injected on the same schedule with equal volumes of i.p. saline. PFC homogenates were prepared, separated on SDS-PAGE gels (30 µg total protein per sample), and probed with anti-AGS3 (1 µg/ml), anti-USP9x (1 µg/ml), and anti-β-Actin (0.1 µg/ml) antibodies. Signal intensities of AGS3 and USP9x were first normalized to that of β-Actin for each sample, and the average normalized ratio of AGS3/Actin (A) or USP9x/Actin (B) for the saline-treated controls was arbitrarily set to 1. Cocaine-induced increases in AGS3 and USP9x are expressed as multiples of the saline-treated average. Both increases were determined to be statistically significant (AGS3 p = 0.0005, USP9x p<0.0001) using a two-tailed, equal-variance Student's t-test.</p

Effects of depleting or overexpressing USP9x on AGS3.

<p>(A) HEK293 cells infected by the lentivirus expressing pLVX-shRNA1, USP9x-shRNA2, or USP9x-shRNA3 were lysed and the lysates were probed with anti-AGS3 (1 µg/ml), anti-GAPDH (0.2 µg/ml), and anti-β-Actin (0.1 µg/ml) antibodies, respectively. The values in the bar graph represent the averages from 6 independent western blot analyses quantified by Li-COR Odyssey Infrared Imaging System (error bars: standard deviations). A representative western blot image was shown. (B) Characterization of AGS3 antibody in immunofluorescence. Images of endogenous AGS3 in HEK293 cells transfected with a non-targeting control siRNA, or one of three different AGS3 siRNAs (siRNA1, 2 or 3; 30 nM, 48 hrs). Cells were then stained using the anti-AGS3 antibody (1 µg/ml). (C–E) Impact of overexpression of an HA-tagged USP9x (C), or the catalytic UCH domain of USP9x (D, E), on the intensity of AGS3 staining 24 hrs after transfection. For (C) and (D), cells overexpressing the relevant proteins are indicated by arrows, arrowheads and asterisks, and the same field is shown under both 20x and 63x magnification in (D). Cells were co-stained with an anti-HA antibody (1∶1000) and the anti-AGS3 (1 µg/ml) antibodies. For (E), the average intensity of AGS3 staining in 50 transfected cells expressing a comparable level of HA-UCH or the catalytically inactive UCH(C/A) mutant was further quantified as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009725#s2" target="_blank">Materials and Methods</a>,” and the value was normalized to that of the non-transfected cells. Scale bar: 20 µm.</p

Impact of overexpression of the UCH domain of USP9x (A, B), or the catalytically inactive UCH(C/A) mutant (C) on the intensity of Gαi3, HSP90 or AGS3 staining 24 hrs after transfection.

<p>Cells overexpressing the relevant proteins are indicated by arrows, arrowheads and asterisks. Cells were co-stained with an anti-HA antibody (1∶1000) (A–C) and anti-Gαi3 (1 µg/ml) (A), anti-HSP90 (5 µg/ml) or anti-AGS3 (1 µg/ml) (C) antibodies. The same field is shown under both 20x and 63x magnification. Scale bar: 20 µm.</p

Mapping of the USP9x-interacting domain of AGS3.

<p>(A) Schematic illustration of the regions covered by the different GST-AGS3 constructs used in the GST pull-down experiments. (B) and (C) Top panels: Coomassie blue gels showed the GST fusion proteins and their relative amounts used in the GST pull-down. The full-length fusions are indicated by asterisks except for GST-TPR whose expression was too low to be detected. The lower molecular weight bands are probably the products of degradation. Bottom panels: Equal amounts of HEK293 cell lysates were incubated with various GST fusion proteins bound to glutathione beads. After wash, the GST pull-down samples were eluted from the beads and probed with anti-USP9x (1 µg/ml) in western blot analysis. (D) The pull-down was performed as described in (C) except that the elutes were probed with an anti-Gαi3 antibody (1 µg/ml).</p

Identification of USP9x as an interacting protein of AGS3.

<p>(A) The SDS-PAGE and mass spectrometric analysis of the immunoprecipitate of EGFP-AGS3 and its associated proteins from Flp-In CV1 stable cells (the top panel). The immunoprecipitate prepared from CV1 cells stably expressing the EGFP alone was included as a negative control. The nature of the doublet bands of EGFP-AGS3 is unclear but presumably caused by protein degradation. Stably expressed EGFP-AGS3, but not EGFP alone, was also able to co-immunoprecipitate Gαi3 as shown by western blotting (the bottom panel). (B) Characterization of the rabbit anti-USP9x antibody in western blotting. The antibody was raised using the N-terminal 500 a.a. of USP9x as an antigen and the crude sera was immunopurified through a peptide column. An equal amount of cell lysates made from HEK293 cells transfected with a non-targeting control siRNA or either of the two USP9x siRNAs (30 nM, 48 hrs) were loaded and probed with the immunopurified antibody (1 µg/ml). GAPDH was used as a loading control and detected with a monoclonal anti-GAPDH antibody (0.2 µg/ml). (C) Characterization of our rabbit anti-USP9x antibody in immunofluorescence. HEK293 Cells were treated with either control or one of the USP9x siRNAs as described above, and stained with the anti-USP9x antibody (1 µg/ml). (D) Co-immunoprecipitation of USP9x with AGS3 from HEK293 cell lysates. Lysates were incubated with an anti-AGS3 antibody (10 µg/ml) or normal rabbit IgG (10 µg/ml, a negative control) and the immunoprecipitates (IP) were probed with either anti-AGS3 (1 µg/ml) or anti-USP9x antibody (1 µg/ml). The heterogenous band pattern of AGS3 has been previously observed and is at least partially caused by the phosphorylation of AGS3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009725#pone.0009725-Groves2" target="_blank">[13]</a>. (E) Co-immunoprecipitation of USP9x with AGS3 from the rat brain PFC lysate. The immunoprecipitation and western blot analysis were conducted as described in (D). (F) Co-immunoprecipitation of AGS3 with USP9x from the rat PFC lysate. The immunoprecipitation and western blot analysis were conducted as described in (D) except that the USP9x antibody (10 µg/ml) was used to immunoprecipitate USP9x and its associated proteins.</p