Fig 1.
Content (A–F) and localization (G–V) of endogenous p41 Ii and MHC II during maturation of DC with TNF-α.
Continuous-line histograms describe the binding of anti-p41 Ii mAb to endogenous p41 Ii. Shadowed histograms represent the corresponding negative controls. Each M1 interval excludes 95% of cells from the corresponding negative control. The percentage of p41 Ii-positive cells in a particular M1 interval and their mean fluorescence intensity (MFI) are stated. A representative analysis of three independent biological repetitions is shown. Confocal images: (H–N) endogenous p41 Ii, (P–V) MHC II (HLA-DR). Bars: 15 μm.
Fig 2.
Colocalization of endogenous p41 Ii and lysosomal cysteine cathepsins S, L and H.
Confocal images show double immunofluorescence and localization of labelled endogenous p41 Ii, cathepsin H, cathepsin S, cathepsin L, CD68, LAMP-2 and HLA-DM in immature DC (A, C) and in mature DC after a 3-day maturation with TNF-α (B, D, E). Fluorescence intensities (A, B) are presented with a rainbow color palette (blue–the lowest intensity, red–the highest intensity). Only merged images are shown elsewhere (C, D, E).
Fig 3.
Active site titration of cathepsin L (2.5 nM) with inhibitory p41 fragment (0.5 nM to 3 nM).
Released fluorescence was measured in duplicate, average values ± SD are shown. (A) Released fluorescence (dF/dt) is related to p41 fragment/cathepsin L molar ratio. (B) SDS-PAGE and (C) IEF of isolated inhibitory p41 fragment (both stained with silver). ST–standards.
Fig 4.
Perinuclear clustering of vesicles in p41 fragment-treated and matured DC.
TEM micrographs: (A) immature DC, (B) mature DC after a 3-day maturation with TNF-α and (C) mature DC, preincubated with 3.5 μM p41 fragment for 6 h prior to their maturation. The positions of nuclei are denoted (*). Bars: 500 nm. Confocal images: (D, F) internalized exogenous p41 fragment (conjugated to Alexa Fluor 488) and (E, G) endogenous cathepsin S (labelled with anti-cathepsin S antibody) in treated immature DC after 6 h (D, E) and in subsequently matured DC after 3 days (F, G). Bars: 15 μm.
Fig 5.
Localization of immunogold labelled p41 Ii (A–B, E–F) and cathepsin S (C–D).
TEM micrographs: immature DC (A, C), mature DC after a 3-day maturation with TNF-α (B, D) and immature DC treated with inhibitory p41 fragment (3.5 μM) for 6 h (E, F). Membranes appear white (non-contrasted). Bars: 200 nm (A–B, D, E–F) and 100 nm (C).
Fig 6.
Effect of internalized p41 fragment on the proteolytic activity of cysteine proteases (A) and the secretion of IL-12/p70 (B, C).
Samples (A): cell lysates of non-treated immature DC, immature DC after a 6-h incubation with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) and non-treated immature DC with 10 μM E-64. Fluorogenic substrate in buffer with DTT was used as blank (BLK). Representative measurements, each of three biological repetitions, are shown. Samples (B and C): cell free supernatants (culture media) of immature DC, preincubated with p41 fragment (0.035 μM, 0.35 μM and 3.5 μM) for 6 h prior to their maturation with TNF-α (B) or LPS (C). Non-treated cells (no preincubation with p41 fragment): immature DC, cultured in the presence of GM-CSF for three days (no maturation), DC matured with TNF-α, and DC matured with LPS. Pretreated but non-matured cells: immature DC, pretreated with 3.5 μM p41 fragment, and cultured in the presence of GM-CSF. IL-12 concentrations (in pg/ml) were measured in triplicate, average values ± SD are shown. (D) Immunolabelled cathepsins L and S in DC lysates. Samples (50 μg per well): (1) immature DC, (2) DC pretreated with 3.5 μM p41 fragment for 6 h, no LPS, (3) DC matured with LPS for 24 h, (4) DC pretreated with 3.5 μM p41 fragment for 6 h and matured with LPS for 24 h.
Fig 7.
Translocation of NF-κB subunit p65 in immature DC treated with p41 fragment.
Confocal images show localization of immunolabelled p65 in differentiated MUTZ-3 cells: (A) non-stimulated, (B) stimulated with LPS, (C) pretreated with 10 μM SN50 and stimulated with LPS, (D) pretreated with 10 μM SN50M and stimulated with LPS, (E) pretreated with 3.5 μM p41 fragment, (F) pretreated with pretreated with 3.5 μM p41 fragment and stimulated with LPS. Nuclei were stained with DAPI. Bars: 10 μm.