c albicans  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: Identification of Cystatin SA as a Novel Inhibitor of Acid Ceramidase *

doi: 10.1074/jbc.M111.260372

Figure Lengend Snippet: The effects of cysSA expression on AC activity and ceramide in cells. a, AC activity was measured in HEK 293T17 cell lysates 24 h after transient expression of the full-length AC cDNA alone or co-transfection of the AC and the cysSA cDNAs. Note that AC activity was significantly lower after co-expression of AC and cysSA as compared with AC only (the asterisk indicates statistical significance, t test, p = 0.004). Expression of the cystatins A, B, C, and E/M cDNAs did not affect AC activity (data not shown). b, after expression of the full-length cysSA cDNA in SK-MEL cells, endogenous AC activity was significantly reduced in the cell lysates 48 h later (p = 0.007). c, ceramide levels also were significantly elevated in the transfected SK-MEL cells at 48 h (p = 0.012). Data represent the mean ± S.E., n = 3 independent experiments.

Article Snippet: To prepare cell lysates, the cell pellets were lysed with the celLytic reagent (Sigma) and centrifuged at 10,000 × g . Inhibition of Cystatin SA with siRNA 200 pmol of cysSA-specific (Santa Cruz Biotechnologies, sc-44521) or control siRNA (Dharmacon, D-001210-02-20) was transfected into SK-melanoma (SK-MEL) cells at 30% confluency in 6-well plates using the RiboJuice TM siRNA Transfection Reagent (EMD Biosciences).

Techniques: Expressing, Activity Assay, Cotransfection, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Identification of Cystatin SA as a Novel Inhibitor of Acid Ceramidase *

doi: 10.1074/jbc.M111.260372

Figure Lengend Snippet: AC and ceramide levels in SK-MEL cells after cysSA siRNA expression. a, AC activity was determined in SK-MEL cell lysates 72 h after transfection with control siRNA (con siRNA), cysSA siRNA, or cysSA siRNA exposed to recombinant cysSA in the culture media (cysSA siRNA + SA). In the latter experiments recombinant cysSA was added to the culture media 48 h after transfection (i.e. cells were exposed to recombinant cysSA for 24 h before being analyzed). Note that AC activity was significantly elevated after cysSA siRNA expression and that this effect was partially (but significantly) mitigated by the addition of recombinant cysSA into the culture media (the asterisk indicates statistical significance, t test, p = 0.003 and p = 0.044, respectively). Data represent the mean ± S.E., n = 3 independent experiments. b, to examine the effect of cysSA siRNA expression on ceramide, the levels were measured 72 h after siRNA transfection ± the inclusion of recombinant cysSA in the media. Note that the amount of ceramide in the cell lysates was significantly decreased after cysSA siRNA transfection, consistent with the elevation of AC activity (p = 0.005). Similarly, the inclusion of recombinant cysSA led to a partial but significant increase in the ceramide level (p = 0.045). c, to confirm the effect of cysSA siRNA expression on the endogenous cysSA mRNA, the levels were quantified by SYBR Green quantitative PCR after transfection with cysSA siRNA or control (con) siRNA. The cysSA mRNA levels were normalized to RPS18 rRNA as an internal control. Bar heights represent the mean values (-fold difference comparing cysSA to SRP18 expression) from three independent experiments. These quantitative PCR findings demonstrated that expression of the cysSA siRNA led to an ∼5-fold reduction of the endogenous cysSA mRNA levels (p = 0.002). d, expression of cysSA protein in the transfected and control cells was detected by Western blotting using a monoclonal antibody specific to cysSA. GAPDH was used as a loading control. Quantification was performed by densitometric scans of the Western blot images. The black bars indicate cysSA; gray bars indicate GAPDH. These results confirmed that in addition to reduction of cysSA mRNA, expression of cysSA siRNA led to reduction of cysSA protein.

Article Snippet: To prepare cell lysates, the cell pellets were lysed with the celLytic reagent (Sigma) and centrifuged at 10,000 × g . Inhibition of Cystatin SA with siRNA 200 pmol of cysSA-specific (Santa Cruz Biotechnologies, sc-44521) or control siRNA (Dharmacon, D-001210-02-20) was transfected into SK-melanoma (SK-MEL) cells at 30% confluency in 6-well plates using the RiboJuice TM siRNA Transfection Reagent (EMD Biosciences).

Techniques: Expressing, Activity Assay, Transfection, Recombinant, SYBR Green Assay, Real-time Polymerase Chain Reaction, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Identification of Cystatin SA as a Novel Inhibitor of Acid Ceramidase *

doi: 10.1074/jbc.M111.260372

Figure Lengend Snippet: Characterization of the AC inhibitory mechanism by cysSA. a, protein lysates were prepared from HEK 293T17 cells transiently transfected with the AC cDNA, alone (−), or in combination with cysSA (+). Cleavage of the AC precursor into the active heterodimer was then assessed by incubation of the lysates at 37 °C at several time points (2, 4, 8, 24, and 48 h). Note that AC precursor cleavage was not affected by co-expression of cysSA at any of the time points. b, AC activity in the transfected HEK 293T17 cell lysates was determined at three different concentrations of substrate (BODIPY C12-ceramide). AC activity at each substrate concentration was significantly reduced (p = 2.8e−05, p = 0.001, p = 1.7e−07, respectively (indicated by the asterisk)) in the presence of cysSA co-transfection. For these experiments equal amounts of cell lysate protein (400 μg) from the transfected cells were incubated in a 100-μl reaction mixture containing BODIPY C12-ceramide. Data represent the mean ± S.E., n = 3 independent experiments. c, shown is a Lineweaver-Burk plot for the data of b, represented by plots y = 0.1509x + 1.7023 (cysSA co-transfection) and y = 0.1069x + 1.2654 (without (w/o) cysSA co-transfection). Interception of the x axes represents −1/Km. The predicted Km values were 0.88 and 0.83 with and without cysSA, respectively. Data represent the mean ± S.E., n = 3 independent experiments. d, shown is a Lineweaver-Burk plot depicting the activity of pure, recombinant AC in the presence of three different concentrations of pure, recombinant cysSA (0.6, 1.2, and 4 μm) at four different concentrations of substrate (NBD C12-ceramide) concentrations (0.067, 0.1, 0.125, and 0.25 μm). e, shown is a Dixon plot (1/V versus [I]), used to predict the Ki through the equation y = 0.5017x + 2.6449. Data represent three independent experiments. AC inhibition was statistically significant for all of the inhibitor concentrations with p values of 0.0006, 1.24e−05, and 2.9e−07 for 0.6, 1.2, and 4 μm cysSA, respectively.

Article Snippet: To prepare cell lysates, the cell pellets were lysed with the celLytic reagent (Sigma) and centrifuged at 10,000 × g . Inhibition of Cystatin SA with siRNA 200 pmol of cysSA-specific (Santa Cruz Biotechnologies, sc-44521) or control siRNA (Dharmacon, D-001210-02-20) was transfected into SK-melanoma (SK-MEL) cells at 30% confluency in 6-well plates using the RiboJuice TM siRNA Transfection Reagent (EMD Biosciences).

Techniques: Transfection, Incubation, Expressing, Activity Assay, Concentration Assay, Cotransfection, Recombinant, Inhibition