Figure 3.
Figure 3. Dex triggers apoptosis in AS-Hsp27-transfected Dex-resistant cells. (A,B) MM.1R cells were transiently transfected with either GFP-tagged AS-Hsp27 (WT) or with empty vector. Following transfections, GFP+ cells were selected by flow cytometry; treated with Dex (5 μM) for 72 hours, and analyzed for apoptosis by annexin V staining (A) and PARP cleavage (B). Median apoptosis was 73% ± 2% in response to Dex plus AS-Hsp27 and 8.1% ± 0.6% in response to Dex plus vector alone (P < .003; n = 3). Cells were also treated with 5 μM Dex for 24 hours and analyzed for apoptosis by PARP cleavage. Lysates were subjected to immunoblot analysis with anti-PARP antibody. Blot shown is representative of 3 independent experiments with similar results. FL indicates full length; CF, cleaved fragment. (C) U266 cells were transiently transfected with either GFP-tagged AS-Hsp27 (WT) or with empty vector. Following transfections, GFP+ cells were selected by flow cytometry; treated with Dex (5 μM) for 72 hours, and analyzed for apoptosis by annexin V staining. Median apoptosis was 74% for Dex plus AS-Hsp27 and 6% for Dex plus vector alone (P < .004; n = 3). (D) Functional specificity of AS-Hsp27 was determined by subjecting protein lysates from AS-Hsp27-transfected or control vector-transfected U266 MM cells to immunoblot analysis with anti-Hsp27 (upper panel) or anti-tubulin antibodies (lower panels). (E) RPMI-8226 cells were transiently transfected with either GFP-tagged AS-Hsp27 (WT) or with empty vector. Following transfections, GFP+ cells were selected by flow cytometry; treated with Dex (5 μM) for 72 hours, and analyzed for apoptosis by annexin V staining. Median apoptosis was 67% for Dex plus AS-Hsp27 and 9% for Dex plus vector alone (P < .005; n = 3). (F) Functional specificity of AS-Hsp27 was determined by subjecting protein lysates from AS-Hsp27-transfected or control vector-transfected U266 MM cells to immunoblot analysis with anti-Hsp27 (upper panel) or anti-tubulin antibodies (lower panel). Error bars in A, C, and E represent standard deviation.

Dex triggers apoptosis in AS-Hsp27-transfected Dex-resistant cells. (A,B) MM.1R cells were transiently transfected with either GFP-tagged AS-Hsp27 (WT) or with empty vector. Following transfections, GFP+ cells were selected by flow cytometry; treated with Dex (5 μM) for 72 hours, and analyzed for apoptosis by annexin V staining (A) and PARP cleavage (B). Median apoptosis was 73% ± 2% in response to Dex plus AS-Hsp27 and 8.1% ± 0.6% in response to Dex plus vector alone (P < .003; n = 3). Cells were also treated with 5 μM Dex for 24 hours and analyzed for apoptosis by PARP cleavage. Lysates were subjected to immunoblot analysis with anti-PARP antibody. Blot shown is representative of 3 independent experiments with similar results. FL indicates full length; CF, cleaved fragment. (C) U266 cells were transiently transfected with either GFP-tagged AS-Hsp27 (WT) or with empty vector. Following transfections, GFP+ cells were selected by flow cytometry; treated with Dex (5 μM) for 72 hours, and analyzed for apoptosis by annexin V staining. Median apoptosis was 74% for Dex plus AS-Hsp27 and 6% for Dex plus vector alone (P < .004; n = 3). (D) Functional specificity of AS-Hsp27 was determined by subjecting protein lysates from AS-Hsp27-transfected or control vector-transfected U266 MM cells to immunoblot analysis with anti-Hsp27 (upper panel) or anti-tubulin antibodies (lower panels). (E) RPMI-8226 cells were transiently transfected with either GFP-tagged AS-Hsp27 (WT) or with empty vector. Following transfections, GFP+ cells were selected by flow cytometry; treated with Dex (5 μM) for 72 hours, and analyzed for apoptosis by annexin V staining. Median apoptosis was 67% for Dex plus AS-Hsp27 and 9% for Dex plus vector alone (P < .005; n = 3). (F) Functional specificity of AS-Hsp27 was determined by subjecting protein lysates from AS-Hsp27-transfected or control vector-transfected U266 MM cells to immunoblot analysis with anti-Hsp27 (upper panel) or anti-tubulin antibodies (lower panel). Error bars in A, C, and E represent standard deviation.

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