Article Figures & Data
Figures
- FIGURE 1.
Three proteins of 27, 53, and 70 kDa elute from the S→ArGranA column with 6 M urea (A). The 27- and 53-kDa proteins were confirmed to be the hsp27 monomer and dimer by tryptic peptide sequencing and immunoblot with hsp27 antisera (B). C, The 70-kDa band in the eluate stains for hsp70 on immunoblot. Lane 1, 6 M urea eluate; lane 2, 1 μg BSA; and lane 3, cytoplasmic lysate of 105 K562 cells.
- FIGURE 2.
A, Hsp27 immunoblot of K562 cytoplasmic extracts precipitated with Ni2+ resin after incubation with nothing (lane 1), pro-rGranA (lane 2), or rGranA (lane 3). K562 (1 × 105 cell equivalents; lane 4) and human LAK cell lysates (1 × 106 cell equivalents; lane 5) were directly loaded without any prior precipitation. Pro-rGranA and rGranA coprecipitate with hsp27. The active enzyme does not cleave it. LAK lysates do not stain for hsp27. B, To verify that hsp27 is not a substrate of granzyme A, K562 cell lysates (105 cell equivalents per lane) were incubated for 1 h at 37°C with nothing (lanes 1and 6) or 560 nM rGranA (lanes 2and 7), S→ArGranA (lanes 3and 8), or pro-rGranA (lanes 4and 9). In lanes 5 and 10, LAK cell lysates (106 cell equivalents) were used as negative control for hsp27 blotting. Electrophoresed samples were analyzed for hsp27 (left) and PHAP II (right) by immunoblot. Only PHAP II was cleaved by the active enzyme.
- FIGURE 3.
A, Flow cytometry of monocytes, CD4, CD8, and B lymphocytes in normal donor PBMC confirms the presence of hsp27 in monocytes (costained with CD14-FITC, upper panel) and the absence of hsp27 in T and B lymphocyte populations (costained with CD4-Cy5, CD8-Cy5, or CD20-FITC, lower panel). The isotype control for Cy5 is not shown, but is similar to the FITC control. B, Western blot with hsp27 antisera of K562 cell lysates obtained either before heat shock or at given times after 1 h of 42°C heat shock. C, Immunoblot of hemopoietic cell lines before (upper panel) or 14 h after heat shock (lower panel) analyzed for hsp27 expression. Lanes 1 to 3 are BLCL lines (PJB-BLCL, DMF-BLCL, 234-BLCL, respectively); lanes 4 and 5 are T cell lines (LAT-T, 603-T); lane 6 is a LAK line (PJB-LAK); lane 7 is K562; lane 8 is HL60; lane 9 is Jurkat; and lane 10, YT-Indy. Each lane contains 106 cell equivalents, except for the lanes with K562 and HL60, which contain 105 equivalents.
- FIGURE 4.
A, Hsp27 translocates to the insoluble fraction of K562 target cells within 10 min of adding CaCl2 to K562-LAK cell 1:1 mixtures to induce granule exocytosis. Hsp27 is detected by blotting with anti-hsp27 mAb after SDS-PAGE analysis of cytoplasmic (C) lysates and insoluble nuclear (N) fractions. When the blot was stripped and reprobed for moesin, there was no change in the cellular localization of moesin during LAK attack. B, To verify that changes in extracellular Ca2+ did not contribute to the translocation of hsp27, control K562 samples treated with 1 mM EGTA for 10 min and then incubated with 5 mM CaCl2 for the indicated times in the absence of LAK cells showed no change in hsp27 localization.
- FIGURE 5.
Immunofluorescence microscopy of Con A-treated COS cells incubated with LAK cells in the presence of EGTA (A) and 20 min after the addition of Ca2+ to induce LAK cell granule exocytosis (B) and stained for hsp27. The LAK cells do not stain for hsp27 and are not visualized. The diffuse cytoplasmic staining of hsp27 in COS cells collapses into long filamentous strands after LAK attack, which concentrate in a perinuclear region. Cells treated with Ca2+, but no LAK cells, show no change in staining (data not shown). When cells are costained for F-actin with rhodamine phalloidin (C–E), the actin cytoskeleton before addition of Ca2+ (C, phalloidin stain) collapses upon addition of Ca2+ to a configuration (D, phalloidin), which colocalizes with that of hsp27 (E, hsp27).
- FIGURE 6.
Hsp27 redistributes from diffuse cytoplasmic staining and coalesces into perinuclear aggregates within 15 min of spinning LAK cells onto Con A-treated COS cell targets. Slides were stained either without added LAK cells (A) or after LAK cells are added (B). The LAK cells are not visible since they do not stain for hsp27.
Tables
Cell Phenotype Mean Fluorescence Intensitya Before heat shock After heat shock CD14+ monocytes 25 117 CD20+ B cells 4 140 CD4+ T cells 4 83 CD8+ T and NK cells 4 58 CD16+ T and NK cells 3 53 -
a Protein expression was analyzed by flow cytometry of stained permeabilized cells before or 14 h after heat shock for 1 h at 42°C. Background staining with an IgG control Ab gave MFI values in the range of 2 to 4 for gated monocytes and lymphocytes before or after heat shock.
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Cell Line Mean Fluorescence Intensitya Before heat shock After heat shock Nonlymphoid cell lines K562 390 585 HL60 94 137 B lymphoblastoid cell lines PJB-BLCL 122 329 DMF-BLCL 118 262 234-BLCL 16 198 T and NK cell lines LAT-T 7 46 603-T 8 44 PJB-LAK 10 97 Jurkat 15 38 YT-Indy 7 8 -
a Protein expression was analyzed by staining permeabilized cells before or 14 h after heat shock for 1 h at 42°C. Background staining with an IgG control Ab gave MFI values in the range of 3 to 5 for each of the cell lines whether before or after heat shock.
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