SUPPLEMENTAL FIGURES
Interferon-γActivates Transglutaminase 2 via aPhosphatidylinositol-3-Kinase
Dependent Pathway: Implications for Celiac SprueTherapy
Thomas R. DiRaimondo, Cornelius Klöck, Chaitan Khosla
Departments of Chemical Engineering (T.R.D., C.K.) and Chemistry (C.K., C.K.)
Stanford University, Stanford CA 94305
Supplemental Figure 1
SupplementalFigure 1: Western blot quantification of IFN-γ induced TG2 expression in T84 monolayers. T84 monolayers treated with0, 10, 100, or 1000 U/mL IFN-γ for 48 h were lysed using RIPA buffer supplemented with 1% (w/v) SDS. Lysates were centrifuged to remove cell debris, and supernatants were analyzed via SDS-PAGE. The protein was transferred to a nitrocellulose membrane, labeled with anti TG2 and anti-β-actin (protein loading control) primary antibodies followed by a HRP conjugated secondary antibody, washed, developed, and scanned using a Typhoon fluorescence imager. (A) Representative scanned image of Western blot. (B) Quantification of TG2 protein levels normalized to the 0 U/mL IFN-γ control. Data shown as average +/- standard error from triplicate measurements.
SupplementalFigure 2
SupplementalFigure 2: Effect of AMPK inhibitor, dorsomorphin, on peptide permeability and TG2 activity in IFN-γ treated T84 monolayers. Permeability of (A) Cy5-33mer and (B) Cy3-D8mer across T84 monolayers treated with IFN-γ for 48 h. At a concentration of 50µM, dorsomorphin is likely toxic to the cells, as indicated by the drastic increase in peptide permeability. (C) TG2 activity, as measured by 5BP incorporation. DMSO levels were kept below 0.1% (v/v) in media. Data shown are normalized to 0U/mL IFN-γ condition represented by mean +/- standard deviation.
SupplementalFigure 3
SupplementalFigure 3: Effectof ROCK inhibitor, Glycyl H-1152, on peptide permeability and TG2 activity in IFN-γ treated T84 monolayers. Permeability of (A) Cy5-33mer and (B) Cy3-D8mer across T84 monolayers treated with IFN-γ for 48 h. (C) TG2 activity, as measured by 5BP incorporation. DMSO levels were kept below 0.1% (v/v) in media. Data shown are normalized to 0U/mL IFN-γ condition represented by mean +/- standard deviation.
SupplementalFigure 4
SupplementalFigure 4: Effect of AKT inhibitor, triciribine, on peptide permeability and TG2 activity in IFN-γ treated T84 monolayers. Permeability of (A) Cy5-33mer and (B) Cy3-D8mer across T84 monolayers treated with IFN-γ for 48 h. At a concentration of 50µM, triciribine is likely toxic to the cells, as indicated by the drastic increase in peptide permeability. (C) TG2 activity, as measured by 5BP incorporation. DMSO levels were kept below 0.1% (v/v) in media. Data shown are normalized to 0U/mL IFN-γ condition represented by mean +/- standard deviation.
SupplementalFigure 5
SupplementalFigure 5: Effect of MLCK inhibitor, PIK, on peptide permeability and TG2 activity in IFN-γ treated T84 monolayers. Permeability of (A) Cy5-33mer and (B) Cy3-D8mer across T84 monolayers treated with IFN-γ for 48 h. (C) TG2 activity, as measured by 5BP incorporation.The buffer used to solubilize the PIK inhibitor did not influence T84 permeability or TG2 activity. Data shown are normalized to 0U/mL IFN-γ condition represented by mean +/- standard deviation.
SupplementalFigure 6
SupplementalFigure 6: Effect of PKC inhibitor, BIM-II, on peptide permeability and TG2 activity in IFN-γ treated T84 monolayers. Permeability of (A) Cy5-33mer and (B) Cy3-D8mer across T84 monolayers treated with IFN-γ for 48 h. (C) TG2 activity, as measured by 5BP incorporation.DMSO used to solubilize BIM-II did not influence T84 permeability. DMSO levels were kept below 0.1% (v/v) in media. Data shown are normalized to 0U/mL IFN-γ condition represented by mean +/- standard deviation.
SupplementalFigure 7
SupplementalFigure 7: Effect of pan-PI3K inhibitor, BEZ235, on peptide permeability and TG2 activity in IFN-γ treated T84 monolayers. Permeability of (A) Cy3-D8mer across T84 monolayers treated with IFN-γ for 48 h. (B) TG2 activity, as measured by 5BP incorporation.DMSO used to solubilize BEZ235 did not influence T84 permeability. DMSO levels were kept below 0.1% (v/v) in media. Data shown are normalized to 0U/mL IFN-γ condition represented by mean +/- standard deviation.
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