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. 2019 Sep 25;11(10):1427. doi: 10.3390/cancers11101427

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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AS1842856 regulates MYB through upregulation of miR-150 (A) AS does not reduce MYB RNA levels. BL cells were treated with AS or DMSO. Treated cells were harvested after 24 h and RNA expression levels were analyzed by qRT-PCR. qRT-PCR data were quantified by the 2^−ΔΔCT method. Data are shown as mean ± SD (n = 3). (B) AS does not induce MYB protein degradation. BL cells were treated with 10 µg/mL CHX and harvested 0–8 h later. Expression of MYB was analyzed by immunoblot. Expression levels of TUBB served as loading control. A representative of 2 independent experiments is shown. Densitometric quantification of MYB/TUBB protein levels was done with ImageJ software (https://imagej.nih.gov/ij/, RRID: SCR_003070). (C) AS induces miR-150 transcription. BL cells were treated with AS or DMSO. Treated cells were harvested after 3 days, followed by miRNA isolation and miRNA cDNA generation as described in Supplemental Methods. miRNA expression levels were analyzed by qRT-PCR. qRT-PCR data were quantified by the 2^−ΔΔCT method. Data are shown as mean ± SD (n = 3). (D–F) BL cell lines were transduced with lentiviral vectors expressing a miR-150 CRISPR/Cas9 knockout construct or a non-targeting control (NT) and FACS sorted. (D) miRNA was isolated and miRNA cDNA was generated 4 days post transduction as described in Supplemental Methods. miRNA expression levels were analyzed by qRT-PCR. qRT-PCR data were quantified by the 2^−ΔΔCT method. Data are shown as mean ± SD (n = 3). (E) miR-150 knockout construct is functional. Genomic DNA was isolated 4 days post transduction and the miR-150 locus was amplified as described in Supplemental Methods (n = 3). (F) miR-150 knockout rescues MYB levels in BL cell lines treated with AS. Cells expressing miR-150sg or NT con were treated with AS for 7 days. Expression of MYB was analyzed by immunoblot. Expression levels of TUBB served as loading control. A representative of 2 independent experiments is shown. Densitometric quantification of MYB/TUBB protein levels was done with ImageJ software (https://imagej.nih.gov/ij/, RRID: SCR_003070).

AS1842856 regulates MYB through upregulation of miR-150 (A) AS does not reduce MYB RNA levels. BL cells were treated with AS or DMSO. Treated cells were harvested after 24 h and RNA expression levels were analyzed by qRT-PCR. qRT-PCR data were quantified by the 2^−ΔΔCT method. Data are shown as mean ± SD (n = 3). (B) AS does not induce MYB protein degradation. BL cells were treated with 10 µg/mL CHX and harvested 0–8 h later. Expression of MYB was analyzed by immunoblot. Expression levels of TUBB served as loading control. A representative of 2 independent experiments is shown. Densitometric quantification of MYB/TUBB protein levels was done with ImageJ software (https://imagej.nih.gov/ij/, RRID: SCR_003070). (C) AS induces miR-150 transcription. BL cells were treated with AS or DMSO. Treated cells were harvested after 3 days, followed by miRNA isolation and miRNA cDNA generation as described in Supplemental Methods. miRNA expression levels were analyzed by qRT-PCR. qRT-PCR data were quantified by the 2^−ΔΔCT method. Data are shown as mean ± SD (n = 3). (D–F) BL cell lines were transduced with lentiviral vectors expressing a miR-150 CRISPR/Cas9 knockout construct or a non-targeting control (NT) and FACS sorted. (D) miRNA was isolated and miRNA cDNA was generated 4 days post transduction as described in Supplemental Methods. miRNA expression levels were analyzed by qRT-PCR. qRT-PCR data were quantified by the 2^−ΔΔCT method. Data are shown as mean ± SD (n = 3). (E) miR-150 knockout construct is functional. Genomic DNA was isolated 4 days post transduction and the miR-150 locus was amplified as described in Supplemental Methods (n = 3). (F) miR-150 knockout rescues MYB levels in BL cell lines treated with AS. Cells expressing miR-150sg or NT con were treated with AS for 7 days. Expression of MYB was analyzed by immunoblot. Expression levels of TUBB served as loading control. A representative of 2 independent experiments is shown. Densitometric quantification of MYB/TUBB protein levels was done with ImageJ software (https://imagej.nih.gov/ij/, RRID: SCR_003070).