<i>miR-375</i> is upregulated in DOX-induced senescent K562 cells.

<p>(A) miRNAs upregulated in 50 nM DOX-treated K562 cells for 4 days as measured by TaqMan® microRNA microarray analysis. The value of the miRNA expression in untreated K562 cells of day 4 is designated 1, and the level of miRNA expression of DOX-treated K562 cells are calibrated to this value. Data represented are the means and SE of 3 independent experiments. (B) Validation of miRNA expression by individual mature TaqMan® microRNA assays using real-time quantitative RT-PCR. The 4 most strongly expressed miRNAs selected from TaqMan® microRNA microarray analysis were further validated. The value of the miRNA expression in untreated K562 cells is designated 1, and the level of miRNA expression of DOX-treated K562 cells of the same day are calibrated to this value. Data represented are the means and SE of 3 independent experiments. (C) Inhibition of <i>has</i>-<i>miR-375</i> by 100 nM <i>has</i>-anti-<i>miR-375</i> inhibitor or 100 nM <i>has</i>-anti-<i>miR-375</i> inhibitor scramble negative control (SC) in K562 cells. After transfection for 48 hours, K562 cells were treated with 50 nM DOX for 5 days. The expression of mature <i>has</i>-<i>miR-375</i> was examined by TaqMan® microRNA assays using real-time quantitative RT-PCR. The value of the <i>has</i>-<i>miR-375</i> expression at day 0 is designated 1, and the levels of all other days of the same treatment are calibrated to this value. Data represented are the means and SE of 5 independent experiments. (D) WST-1 assay was performed to determine cell proliferation after 100 nM <i>has-</i>anti-<i>miR-375</i> inhibitor or 100 nM <i>has</i>-anti-<i>miR-375</i> inhibitor SC transfection followed by 50 nM DOX treatment in K562 cells. Data represented are the means and SE of 5 independent experiments. *Indicates significant difference compared to cells treated with 50 nM DOX and treated with 100 nM anti<i>-miR-375</i> SC and 50 nM DOX (<i>p</i><0.05). (E) Overexpression of <i>miR-375</i> by 100 nM <i>has</i>-<i>miR-375</i> precursor or 100 nM <i>has</i>-<i>miR-375</i> precursor SC in K562 cells. The measurement and calculation of mature <i>has</i>-<i>miR-375</i> expression were as described in (C). Data represented are the means and SE of 5 independent experiments. (F) WST-1 assay was performed to determine cell proliferation after 100 nM <i>has</i>-<i>miR-375</i> precursor or 100 nM <i>has</i>-<i>miR-375</i> precursor SC transfection in K562 cells. Data represented are the means and SE of 5 independent experiments. *Indicates significant difference compared to both untreated K562 cells and cells treated with 100 nM <i>has</i>-<i>miR-375</i> precursor SC (<i>p</i><0.05).</p

mRNA expression of senescence-associated genes in K562 cells treated with 50 nM DOX as measured by real-time quantitative RT-PCR.

<p>The x-axis indicates the days post DOX treatment and the y-axis represents the relative mRNA expression level. The value of the mRNA expression at day 0 is designated 1, and the levels of all other days are calibrated to this value. Data represented are the means and SE of 5 independent experiments.</p

Expression of putative <i>miR-375</i> target genes in DOX-induced senescent K562 cells.

<p>(A) mRNA expression of putative <i>miR-375</i> target genes in K562 cells treated with 50 nM DOX for 3 and 4 days as measured by real-time quantitative RT-PCR. The value of the mRNA expression in untreated K562 cells of the same day is designated 1, and the level of mRNA expression of DOX-treated K562 cells are calibrated to this value. Data represented are the means and SE of 5 independent experiments. (B) Expression of <i>14-3-3zeta</i>, <i>LDHB</i>, and <i>SP1</i> genes in K562 cells treated with 50 nM DOX (<i>DOX</i>) transfected with 100 nM <i>has-</i>anti-<i>miR-375</i> inhibitor followed by 50 nM DOX treatment (<i>Inh</i>) or transfected with 100 nM <i>has</i>-<i>miR-375</i> precursor (<i>Pre</i>) for 3 and 4 days. The calculation of gene expression was as described in (A). Data represented are the means and SE of 3 independent experiments.</p

DOX induced senescence but PTX not senescence in K562 cells.

<p>(A) K562 cells treated with 50 nM of DOX for 4 days were stained for SA-β-gal activity followed by DAPI staining. Original magnification is 400×. Representative microscopic fields are shown. (B) K562 cells were treated with 50 nM of DOX for 5 days, and the percentages of apoptotic cells were determined by Annexin V/PI staining followed by flow cytometric analysis. Data represented are the means and SE of 3 independent experiments. (C) K562 cells were treated with 50 nM of DOX for 5 days, and DNA contents were measured by flow cytometric analysis after PI staining. Data represented are the means and SE of 3 independent experiments.</p