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. Author manuscript; available in PMC: 2008 Aug 14.
Published in final edited form as: Cancer Res. 2006 Feb 1;66(3):1702–1711. doi: 10.1158/0008-5472.CAN-05-3092

Figure 4. Functional role for BiP and PERK in dormant cell drug resistance.

Figure 4

A, Western blot analysis of siRNA-mediated down-regulation of BiP in D-HEp3 cells. siRNA to BiP caused a >3-fold down-regulation in BiP protein; no change was observed on GAPDH levels except when an siRNA to this gene was used, which did not affect BiP expression levels. B and C, control and BiP siRNA–transfected cells were treated with or without 5 μg/mL doxorubicin (Dox) for 24 hours and analyzed for overall cell death by trypan blue exclusion test (B) or by TUNEL assay (C). B, columns, mean; bars, SD. *, P < 0.01. C, % cells in each quadrant. PMT4, PI staining; PMT2, FITC-dUTP staining. Note the increase in the percentage of apoptotic cells in the upper right quadrant following BiP-si and doxorubicin treatment. D, quantitation of DNA breaks as measured using the TUNEL assay Apo-Direct kit. Control and BiP siRNA–transfected cells treated with or without 2 μg/mL (T-HEp3) or 5 μg/mL (D-HEp3) of doxorubicin were analyzed for apoptosis by TUNEL assay as in (C). Columns, mean of % FITC-dUTP–positive cells in each sample; bars, SD. E, representative fields of D-HEp3 cells transiently cotransfected with EGFP and empty vector, wt-PERK, PERK-ΔC, or GADD34, respectively. After transfection (48 hours), cells were treated overnight with doxorubicin, and then cells were fixed in 70% ethanol and stained with Hoescht 33342 (20 mmol/L). Nonspecific nuclear fluorescence due to doxorubicin accumulation in cells crossed to the GFP channel. Bar, 60 μm. Open arrowheads, left, GFP-negative apoptotic cells. Arrow, GFP-positive nonapoptotic cells. Closed arrowhead, right, GFP-positive apoptotic cells. F, quantitation of apoptotic bodies as measured using Hoescht 33342 in (E). At least 150 cells positive for GFP were scored for the presence of apoptotic bodies characterized by nuclear DNA condensation. Columns, mean; bars, SD (E). #, P < 0.05, EGFP + doxorubicin versus GADD34 + doxorubicin. *, P < 0.01, EGFP + doxorubicin versus PERK-ΔC + doxorubicin, as determined by ANOVA test. G, representative fields of D-HEp3 cells transiently cotransfected with EGFP and empty vector, PERK-ΔC, or GADD34 vectors, respectively. After transfection (48 hours), cells were treated overnight with etoposide and were fixed and stained with α-cleaved caspase-3 antobody (see Materials and Methods) and analyzed for caspase-3 activation by immunofluorescence. Arrows, b and f, EGFP-negative and caspase-3–positive cells. Arrows, d and h, EGFP-positive and caspase-3–positive cells. Bar, 40 μm. H, quantitation of caspase-3 activation as measured using immunofluorescence in (G). D-HEp3 cells transiently cotransfected with EGFP and PERK-ΔC or GADD34, respectively, were treated, fixed, and stained as described in Materials and Methods. More than two hundred cells positive for GFP were scored for caspase-3 activation. Columns, mean; bars, SD. #, P < 0.001, GADD34 versus GADD34 + etoposide or PERK-ΔC versus PERK-ΔC + etoposide. *, P < 0.005, EGFP + etoposide versus GADD34 + etoposide or PERK-ΔC + etoposide as determined by Mann-Whitney test.