Fig 2: Subcellular localization of the C316A, C319S and R569W MPO mutants stably expressed in T47D cells.Cells grown on glass coverslips were fixed, permeabilized and double-labeled with polyclonal MPO antibodies and the indicated organelle marker antibody. Mounted coverslips were imaged with a 63X objective on a Zeiss LSM 710 confocal microscope. (A) T47D-C316A cells labeled with antibodies against MPO and Lamp1 and shows that the C316A mutant accumulates normally within lysosomes. (B) T47D-C319S cells labeled with antibodies against MPO (all panels) and Lamp1 (top panel), the ER marker KDEL (second panel), the trans-Golgi marker RCAS1 (third panel) and the cis-Golgi marker GM130 (fourth panel). Distinct localizations of the C319S mutant to both the ER (arrow) and Golgi (arrowhead) are indicated on the second panel. (C) T47D-R569W cells labeled with antibodies against MPO and the ER marker protein disulfide isomerase (PDI).

Fig 3: Comparison of constitutively secreted MPO precursors from recombinant cell lines, HL60 cells, and human serum.(A) Secreted MPO species were partially purified from the conditioned media of MPO-expressing cell lines on SP-sepharose and analyzed by immunoblot with a multi-epitope MPO polyclonal antibody (upper blots) or anti-peptide antibody specific for the pro-domain of MPO (lower blots). ProMPO derived from T47D-MPO cell extracts is loaded in the far left lane for comparison (left-hand blots). The nature of the observed immunoreactive bands was further examined by immunoblot analysis of secreted MPO species from HL60 and T47D-MPO cells after complete deglycosylation with PNGaseF to more accurately compare gel mobility and domain specific immunoreactivity (right-hand blots). (B) Immunoblot analysis of MPO species SP-purified and immunoprecipitated from human serum. Three species of MPO are observed in human serum and are defined based on relative mobility and domain-specific immunoreactivity in comparison with cellular and secreted MPO species derived from the T47D-MPO cell line. A reference line is included between glycosylated proMPO and the”cleaved” proMPO intermediate for mobility comparison between left and right blots. The bottom of the right-hand blot is truncated because of strong cross-reactivity of secondary antibodies with the immunoprecipitating antibody in that region. (C) Secreted MPO was partially purified on SP-sepharose and then assayed for peroxidase activity and total MPO content and the relative specific activity (Active MPO/Total MPO) determined as described in “Materials and Methods”. (D) mRNA expression profile of catalase from 59 cancer cell lines plotted in chromatogram format. The data were obtained from the NCI-60 public database and show that HL60 cells express high levels of catalase, exceeding the next highest cell line by a full order of magnitude. (E) Specific peroxidase activity of cellular MPO (cell extract) and secreted MPO (media) derived from HL60 and T47D-MPO cells grown in the absence or presence of catalase for 48 hrs. Arrows indicate the catalase-dependent increase in specific activity observed for secreted MPO, but not cellular MPO, for each cell line. (C & E) Assay points are triplicate measurements and plotted as mean ± SE. The data shown is representative of at least three independent experiments.

Fig 4: Effect of cathepsin inhibitors on the processing, activity and endocytic trafficking in T47D-MPO cells.(A) T47D-MPO cells were grown with or without 8 µM ALLN for 44 h. MPO was partially purified from the resulting cell extracts on SP-sepharose and analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody to detect all MPO species (left panel) or antibodies specific for epitopes within the light chain of MPO (right panel). (B) T47D-MPO cells were grown in the presence of the indicated concentrations of e64d for 44 h and the resulting cell extracts analyzed by immunoblot under non-reducing (-DTT) and reducing (+DTT) conditions with multi-epitope polyclonal MPO antibody. (C) Samples analyzed in panel A were assayed for peroxidase activity and total MPO content and the relative specific activity (Active MPO/Total MPO) determined as described in “Methods and Materials”. (D) T47D-MPO cells were grown on glass coverslips in the presence of 8 µM ALLN for 72 h. The treated cells were fixed, permeabolized and double-labeled with antibodies against MPO (red) and Lamp1 (green). Mounted coverslips were imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope.

Fig 5: Subcellular localization of MPO in cells that DO (T47D) and DO NOT (BT549) produce heterotetramer.Cells grown on uncoated glass coverslips were fixed, permeabolized and double-labeled with antibodies against the indicated proteins. Mounted coverslips were imaged with a 63x oil objective on a Zeiss LSM 710 confocal microscope. (A) Fluorescent images of the T47D-MPO stable cell line showing that MPO colocalizes with the lysosomal membrane protein Lamp1, but not with the early endosome protein EEA1 or the cis-Golgi protein GM130. An extra inset of a cell with very large lysosomes is included to more easily visualize that MPO-immunoreactivity (red) is concentrated within the lumen of the lysosomes. (B) Fluorescent images of the BT549-MPO stable cell line showing that MPO does not colocalize with the lysosome marker Lamp1 in these cells, but, the punctate staining pattern does partially co-localize with the endoplasmic reticulum marker KDEL. (n) nucleus.