CCN3 mRNA and protein is down-regulated in primary cells from patients with CML, and expression increases upon entering remission. (A) RNA was extracted from bone marrow samples taken from patients with CML at diagnosis and following treatment (patient 1 hematopoietic response, HR; patients 2 and 3 complete cytogenetic response, CCR), and real-time PCR was performed to determine CCN3 and BCR-ABL mRNA levels. Results are presented as the fold change in BCR-ABL () and CCN3 (□) expression following treatment (mean of 3 determinations ± SD). (B) Protein lysates extracted from CML patient bone marrow samples taken at diagnosis (“D”) and following response to treatment (“R”) were subjected to Western blot analysis to identify CCN3 protein expression. Three normal bone marrow samples are included for comparison. Patient 1, HR; patients 2 and 3, CCR. (C) CD34⁺ cells were extracted from the bone marrow of 3 patients with CML at diagnosis and treated for 72 hours in vitro with imatinib (1 μM). Western blot analysis of the medium in which the cells were grown and protein cell lysates was performed to identify CCN3 protein. Optical densitometry was performed on the Western blot. The mean signal from NBM was assigned 100% and measurements were expressed as a percentage compared to the signal from NBM. (D) Optical densitometry was performed on the Western blot from panel C. CCN3 expression in CD34⁺ cells grown without imatinib were assigned 100%. CCN3 expression in CD34⁺ cells treated with imatinib for 72 hours was expressed as a percentage compared with cells that were not treated. (E) Confocal microscopy was used to detect CCN3 expression (green fluorescence) in mononuclear cells from a patient with CML at diagnosis (i) and following a complete cytogenetic response (ii). Images were collected using a Bio-Rad Microradiance confocal laser scanning microscope with an oil immersion lens (magnification, × 80). Propidium iodide was used to stain the nuclei. (F) Confocal microscopy was used to detect CCN3 protein (green fluorescence) in CD34⁺ cells from a patient with CML at diagnosis (i), normal bone marrow (ii), and a patient with thrombocythemia (iii). Images were collected using a Bio-Rad Microradiance confocal laser scanning microscope with an oil immersion lens (magnification, × 110). Propidium iodide was used to stain the nuclei.