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Blood, Vol. 107, Issue 10, 4003-4010, May 15, 2006
Bcr-Abl reduces endoplasmic reticulum releasable calcium levels by a Bcl-2–independent mechanism and inhibits calcium-dependent apoptotic signaling
Blood Piwocka et al.
107: 4003
Supplemental materials for: Piwocka et al
Files in this Data Supplement:
- Figure S1. Bcr-Abl expression is associated with endoplasmic reticulum stress symptoms (JPG, 34 KB)
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(A) Western blot analysis of Bcr-Abl protein levels and (B) Grp78 protein level in untreated 32D, C2 and C4 cells. Antibodies were used as follows: anti-c-abl (Ab-3) (Calbiochem), anti-Grp78 (StressGen Biotechnologies, York, UK), anti— -actin clone AC-15 (Sigma-Aldrich). Cells were lysed in modified RIPA buffer as described previously16. Equal amounts of protein were resolved using SDS-PAGE and electrotransferred to nitrocellulose. All secondary antibodies were horseradish peroxidase conjugated (DakoCytomation Ireland Ltd, Galway, Ireland) and detected using Enhanced Chemiluminescence System (ECL, Amersham, Little Chalfont, UK). -actin was used as an internal loading control. Representative data are shown. (C) Semi quantitative RT-PCR analysis of CHOP gene expression in untreated 32D, C2 and C4 cells. Total RNA was extracted from 5—10×106 cells using TRI-Reagent (Molecular Research Center Inc., Cincinnati, OH, USA). Total RNA was converted to cDNA using M-MLV Reverse Transcriptase (Promega UK Ltd Southampton, UK). Primers for PCR were: CHOP forward: 5’-CAC CTA TAT CTC ATC CCC AGG AA-3’; CHOP reverse: 5’-CCG CTC GTT CTC TTC AGC TA-3’; actin forward: 5’-GAC CCA GAT CAT GTT TGA GA-3’, actin reverse: 5’-CTT CAT GAG GTA GTC TGT CA-3’. Detection of -actin cDNA was used as a loading control.
- Figure S2. Rhod-2 specifically stains mitochondria, and fluorescence is consistently higher in 32D cells treated with VP16 (JPG, 56.8 KB)
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(A) Rhod-2 specific mitochondrial localization determined by confocal microscopy. 32D untreated cells were dual labeled with 1 µM Rhod-2 and 200 nM MitoTracker Green, a mitochondrial marker, After three washes with PBS, cells were observed by confocal microscopy (Leica Microsystems Heidelberg GmbH). Cells were illuminated by the 488 nm and 543 nm emission lines of a krypton/argon laser and fluorescence was detected simultaneously using FITC filter for MitoTracker Green detection and TRITC filter for Rhod-2 detection. Separate staining with Mitotracker (left), Rhod-2 (center) as well as merged picture (overlay, right) are shown. Rhod-2 and MitoTracker Green are co-localized in most intracellular structures (overlay). The only notable exception in co-localization is the result of Rhod-2 labeling of the nucleolus as previously reported46. Rhod-2 labeling of mitochondria (arrowhead) is easily distinguished from labeling of the nucleolus (arrow). Representative slides are presented.
(B) TILL photonic analysis of Rhod-2 fluorescence levels after 14 h of VP-16 treatment. 32D, C2 and C4 cells untreated (panel 1), or VP-16 treated (panel 2) were loaded with Rhod-2 and digital imaging was performed using TILL Photonics GmbH system in order to quantify levels of Rhod-2 staining in single cells. Cells were illuminated with the Polychrome IV monochromator (TILL photonics) at 545 nm. Emission filter used for Rhod-2 fluorescence detection was U-MWIG2 Olympus (Ex. 520-550, Em.589IF, dichromatic mirror 565). Fluorescence emissions were captured by CCD camera (Imago, TILL photonics) attached to a 12-bit frame grabber and analyzed using TILLvisION 4.01 acquisition and analysis software. Images were captured with an exposure time of 150 ms in five different fields for each slide. Fluorescence is shown in an artificial color scale, where blue represents low fluorescence and therefore low levels of mitochondrial calcium and red represents high levels of fluorescence and therefore high mitochondrial calcium. Cells with high levels of mitochondrial calcium were only visible in the 32D cells, whereas in the Bcr-Abl expressing cells, almost no increase in fluorescence was detected. VP-16 treated cells were additionally treated with 1 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (panel 3), which depolarizes mitochondria and effectively inhibits their ability to take up calcium. As shown in Fig S2B panel 3, an immediate drop of fluorescence occurred in all three cell lines, indicating that Rhod-2 specifically stained calcium localized in the mitochondria and not in the cytosol. Representative pictures from three separate experiments are shown.
(C) Statistical analysis of Rhod-2 fluorescence level elevation after 14 h of VP-16 treatment. For each case at least 150 cells were analyzed in three separate experiments and mean signal was calculated on the basis of mean fluorescence of the captured area of a single cell masked and detected using TILLvisION software. For each of three experiments mean basal fluorescence (F0) for untreated cells and then the mean elevated fluorescence (F) for VP-16 treated cells were calculated. Changes in the mitochondrial calcium were represented as %ΔF/F0 ratio; where ΔF/F0 = 100((F- F0)/F0). Finally, %ΔF/F0 ratios from all experiments were statistically analyzed and presented as mean ± S.D. (mean %ΔF/F0). This data indicates that 32D cells exhibit significantly higher (almost 3 fold higher), mitochondrial calcium, following drug treatment compared to Bcr-Abl expressing cells.
- Figure S3. Calpains are activated more strongly in 32D cells than in Bcr-Abl expressing C2 cells after VP-16 treatment (JPG, 31.2 KB)
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(A) 32D and C2 cells were treated for 16 h with VP-16. Calpain activity assays were carried out according to the instructions of the manufacturer (Calbiochem). The assay is based on fluorometric detection of cleavage of the substrate Ac-LLY-AFC. Basal fluorescence levels of untreated were considered as 100%, and treated cell samples were expressed relative to basal levels. Results are shown as mean +/— S.D. of 3 independent experiments. UNT: untreated samples; VP-16: cells treated with VP-16; Control: fluorescent calpain substrate in buffer without cell lysates. A 2-fold increase in activity was determined in VP-16 treated compared to untreated C2 cells, whereas in 32D cells, calpain activity was increased to up to 5-fold, in drug treated cells.
(B) Detection of m-calpain (anti-M-calpain/calpain2 antibody, Calbiochem) by Western blotting: 32D and C2 cells were treated for 18 h with VP-16 (7.5 µg/ml), lysed and subjected to SDS-PAGE followed by Western blotting. -actin was used as a loading control. Blot is representative of three independent experiments. Calpains exist in the cytosol as an inactive 80 kDa pro-enzyme which is cleaved under activating conditions. The 80 kDa form of m-calpain was not detected in 32D cells treated with VP-16 indicating strong calpain activation in these cells, whereas in C2 cells the 80 kDa band level was only slightly decreased indicating that calpain activity is higher in 32D cells compared to C2 cells.
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