American Society of Hematology

Figure 3.

$Expansion of BCR-ABL+senescent MgkL cells expressing TGF-β in BM of CML mouse. (A) Immunohistochemical staining of BM obtained from CML or untreated mice. BMs were obtained from LIC recipients 3 weeks after the transplantation and subjected to immunohistochemical analysis using anti-p16 (upper) or anti-p21 antibodies (lower). Representative results from 5 individual experiments are shown. Arrows indicate the cells which were morphologically identified as megakaryocytes. Original magnification, ×200. Insets indicate high-power magnification images (×400). Scale bars, 50 μm. (B) CD150+CD41+ MgkL cells in the BM from LIC recipient mice or the mice transplanted with control GFP-transduced KLS+ cells. BMs were obtained from WT- or DKO-derived LIC recipients or the mice transplanted with control GFP-transduced KLS+ cells at the indicated time intervals after the transplantation. Flow cytometric analysis was conducted. Upper panels show representative results obtained from 4 WT-derived LIC recipients 3 weeks after the transplantation. Lower panels show the proportion (left) and absolute numbers (right) of CD150+CD41+ MgkL cells in BCR-ABL+ or BCR-ABL− populations (n = 4). (C) Expression of senescence-associated and SASP-related molecules. BM cells were obtained from WT-derived LIC recipients 3 weeks after the transplantation and fractionated according to expression level of BCR-ABL and surface markers. Total RNA was extracted from the obtained cell fractions and subjected into qRT-PCR to determine senescence-associated (upper) and SASP-related molecules (lower) (n = 4 or 5). The expression level was calculated relative to Gapdh gene and normalized to the averaged value of BCR-ABL− total cells. (D) Protein expression of intracellular p16 and intranuclear p21. Mean fluorescence intensities (MFIs) were determined in each fraction (n = 4). (E) Intracellular TGF-β1 expression. MFIs were determined for each fraction (n = 6). Data are presented as the mean ± SD. Statistical significance was evaluated using 1-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test. *P < .05; **P < .01.$

Expansion of BCR-ABL⁺senescent MgkL cells expressing TGF-β in BM of CML mouse. (A) Immunohistochemical staining of BM obtained from CML or untreated mice. BMs were obtained from LIC recipients 3 weeks after the transplantation and subjected to immunohistochemical analysis using anti-p16 (upper) or anti-p21 antibodies (lower). Representative results from 5 individual experiments are shown. Arrows indicate the cells which were morphologically identified as megakaryocytes. Original magnification, ×200. Insets indicate high-power magnification images (×400). Scale bars, 50 μm. (B) CD150⁺CD41⁺ MgkL cells in the BM from LIC recipient mice or the mice transplanted with control GFP-transduced KLS⁺ cells. BMs were obtained from WT- or DKO-derived LIC recipients or the mice transplanted with control GFP-transduced KLS⁺ cells at the indicated time intervals after the transplantation. Flow cytometric analysis was conducted. Upper panels show representative results obtained from 4 WT-derived LIC recipients 3 weeks after the transplantation. Lower panels show the proportion (left) and absolute numbers (right) of CD150⁺CD41⁺ MgkL cells in BCR-ABL⁺ or BCR-ABL⁻ populations (n = 4). (C) Expression of senescence-associated and SASP-related molecules. BM cells were obtained from WT-derived LIC recipients 3 weeks after the transplantation and fractionated according to expression level of BCR-ABL and surface markers. Total RNA was extracted from the obtained cell fractions and subjected into qRT-PCR to determine senescence-associated (upper) and SASP-related molecules (lower) (n = 4 or 5). The expression level was calculated relative to Gapdh gene and normalized to the averaged value of BCR-ABL⁻ total cells. (D) Protein expression of intracellular p16 and intranuclear p21. Mean fluorescence intensities (MFIs) were determined in each fraction (n = 4). (E) Intracellular TGF-β1 expression. MFIs were determined for each fraction (n = 6). Data are presented as the mean ± SD. Statistical significance was evaluated using 1-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test. *P < .05; **P < .01.

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