Supplementary Materials and Methods
Ethic statement
The mouse experimental design and protocols used in this study were approved by “the regulation of the Institute of Microbiology, Chinese Academy of Sciences of
Research Ethics Committee” (Permit Number: PZIMCAS2012001). All mouse experimental procedures were performed in accordance with the Regulations for the
Administration of Affairs Concerning Experimental Animals approved by the State
Council of China. For the use of clinical materials for research purposes, prior patients’ consent and approval were obtained from the Center of Oncology and
Hematology, the First Affiliated Hospital of Guangzhou Medical University, China.
Antibodies and reagents
The following antibodies were used in this study: FLAG (Sigma F1804 M2), c-Myc (Santa Cruz sc-40 9E10), Bcr (Santa Cruz sc-885 N-20), Bcl-xL (Cell
Signaling 2764 54H6), STAT5 (Cell Signaling 9358 3H7), phospho-STAT5 (Tyr694)
(Cell Signaling 9359 C11C5), PTEN (Cell Signaling 9559 138G6), AKT (Cell
Signaling 2967 2H10), and phospho-AKT (Ser473) (Cell Signaling 4060 D9E). DNA methyltransferase inhibitor 5-Aza-2-deoxycytidine (5-Aza-dc) was purchased from
Sigma.
DNA construction
LncRNA-BGL3 was subcloned into the two-promoter-based lentiviral vector
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pll3.7 to generate pll3.7-LncRNA-BGL3-GFP in which the transcription of lncRNA-BGL3 and GFP was initiated using two independent promoters (EF-1a and
CMV respectively). LncRNA-BGL3 mutant devoid of the specific miRNAs binding sites, in which seed matches of lncRNA-BGL3/PTEN-targeting miRNAs were mutagenized from ‘GCACTTT’ to ‘CCTCATA’, was generated using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, USA). LncRNA-BGL3 was subcloned into pMIG-IRES-GFP to generate pMIG-LncRNA-BGL3-GFP. Bcr-Abl was subcloned into pll3.7 to generate pll3.7-Bcr-Abl-GFP. LncRNA-BGL3 was subcloned into pll3.7-Bcr-Abl-GFP to generate pll3.7-Bcr-Abl-LncRNA-BGL3. Short hairpin RNA (shRNA)-expressing constructs were generated by subcloning shRNA sequences into BamH
Ⅰ and EcoR
Ⅰ sites of pSIH-H1-GFP vector (System
Biosciences). The shRNA sequences targeting Bcr-Abl are
5’-GCAGAGTTCAAAAGCCCTT-3’
(sh-Bcr-Abl),
5’-TAAGCAGAGTTCAAAAGCC-3’ (sh-Bcr-Abl-1), and
5’-GAGTTCAAAAGCCCTTCAG-3’ (sh-Bcr-Abl-2). shRNA sequence targeting lncRNA-BGL3 is 5’-CTCTGCTCTTAAAGATAATTT-3’, shRNA sequence targeting
PTEN is 5’-AAGATCTTGACCAATGGCTAA-3’.
Cellular fractionation
K562 cells were collected, rinsed twice in PBS, and resuspended in lysis buffer
(10 mM Tris [pH 8.4], 140 mM NaCl, 1.5 mM MgCl2, 0.5 % Nonidet P-40, 1 mM dithiothreitol, RNasin). The supernatant was saved as the cytoplasmic fraction after
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centrifuging at 1,000 g for 3 minutes. Nuclear pellets were resuspended in lysis buffer.
One-tenth volume of the detergent (3.3% sodium deoxycholate, 6.6% Tween 40) was added. Nuclei were pelleted by centrifugation at 1,000 g for 3 min, and the supernatant was added to the cytoplasmic fraction. Together, these are considered the cytoplasmic fraction. The nuclei were rinsed once in lysis buffer. RNAs extracted from each of the fractions and from un-fractionated cells were subjected to following
RT-PCR analysis of the levels of nuclear control transcript (U6), cytoplasmic control transcript (GAPDH), and lncRNA-BGL3.
RNA immunoprecipitation (RIP)
RIP experiments were performed using the Magna RIP RNA-Binding Protein
Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. For Ago2 RIP, rabbit anti-Ago2 antibody (Cell Signaling 2897 C34C6) was used.
RT-PCR, quantitative real-time PCR, and Western blotting
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA).
First-strand cDNA was generated using M-MLV Reverse Transcriptase (Promega,
Madison, WI) and oligo (dT) primers (Takara, Dalian, P.R. China). The following
PCR was performed using rTaq DNA Polymerase and quantitative PCR using
SYBR® Premix Ex Taq™ II (Takara, Dalian, P.R. China) with gene specific primers.
Expression level of β-actin was used as a control. For miRNA quantitative real-time
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PCR, the E. coli poly (A) polymerase was used to add poly (A) tail to total RNA.
Complementary DNA was synthesized using the HiFi-MMLV reverse transcriptase and specific reverse transcription primers (CWBio. Co., Ltd. Beijing, China). SYBR
Green uptake in double-stranded DNA was measured using UltraSYBR mixture
(CWBio. Co., Ltd. Beijing, China). Quantitative PCR was performed using the
Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City,
USA).
For Western blotting, cells were treated as indicated in figure legends, and the harvested cells were lysed for protein collection. Samples were separated on
SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with antibodies as indicated. Where indicated, Western blot signals were quantified by densitometry.
Statistical analysis
Results were expressed as mean values ± standard error (mean ± SE). Statistical analysis was performed by Student’s t-test. A level of P < 0.05 was considered to be significant.
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Supplementary Figure Legends
Figure S1. LncRNA expression in K562 cells after disruption of Bcr-Abl expression or inactivation of Abl kinase. (a) RT-PCR analysis of expression of selected lncRNAs in K562 cells expressing Bcr-Abl shRNA (left panel), or treated with imatinib (10

M) for 24 h (right panel). Shown are representative data from three independent experiments with similar results. (b) The protein-coding potential analysis of lncRNA-BGL3 was performed using ORF finder from NCBI.
Figure S2. Expression of lncRNA-BGL3 in K562 cells is downregulated by
Bcr-Abl, and lncRNA-BGL3 overexpression sensitizes v-Abl-transformed cells to undergo apoptosis. (a) Nucleotide resolution of Bcr-Abl shRNAs sequences in
Bcr-Abl. As indicated, sh-Bcr-Abl-1 and sh-Bcr-Abl-2 shifted three nucleotides to the
Bcr or to the Abl gene respectively as compared sh-Bcr-Abl.
All these shRNAs target
Bcr-Abl fusion point.
(b and c) LncRNA-BGL3 expression in K562 cells treated in a time course with imatinib (10

M) was detected by RT-PCR and quantitative real-time PCR analysis. Results in (c) are presented as mean ± SEM ( n = 3), * P <
0.05. (d and e) RT-PCR and quantitative real-time PCR analysis of expression of lncRNA-BGL3 in K562 cells stimulated with varying concentrations of imatinib for
24 h. Results in (e) are presented as mean ± SEM ( n = 3), * P < 0.05.
(f and g)
RT-PCR was performed to examine the expression of lncRNA-BGL3 in lncRNA-BGL3 overexpressing, or lncRNA-BGL3 knockdown K562 cells treated with (g) or without imatinib (f). (h) Cell viability of K562 cells overexpressing
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lncRNA-BGL3 or empty vector (EV) untreated with imatinib was assayed by flow cytometry. Results are presented as mean ± SEM ( n = 3). (i) Relative absorbance value of K562 cells expressing lncRNA-BGL3 or EV in the absence of imatinib was determined by MTT assay. The cells expressing lncRNA-BGL3 or EV were plated on the 96-well plate at 10000 cells per well. MTT was added after culture for 72 h, and the absorbance value of each well at 590 nm was measured. The average absorbance value of EV cells is 1. Results are presented as mean ± SEM (n = 3).
(j) RT-PCR analysis of lncRNA-BGL3 expression in v-Abl-transformed cells expressing lncRNA-BGL3 or EV. (k) LncRNA-BGL3 levels in (j) were quantitated by densitometry, and normalized to actin levels. The average value in EV is 1. Plotted are the average levels from three independent experiments. The error bars represent the SEM ( n = 3). (l) Survival of v-Abl-transformed cells stably expressing lncRNA-BGL3 or EV was analyzed by flow cytometry after treatment with imatinib.
Results are presented as mean ± SEM ( n = 3). * P < 0.05.
Figure S3. Enhanced lncRNA-BGL3 expression inhibits tumor formation induced by K562 leukemic cells in xenograft mouse model.
(a) Nude mice were subcutaneously injected with K562 cells expressing lncRNA-BGL3 (lncRNA-BGL3),
Bcr-Abl shRNA (sh-Bcr-Abl), or empty vector (EV). The tumor volumes were measured at indicated time points. Plotted are results from three independent experiments. Error bars represent SE. n = 10. * P < 0.05. (b) Tumors were excised from mice. Shown are representative images from three independent experiments with
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similar results. (c) Relative volume of tumors excised from nude mice injected with
K562 cells expressing lncRNA-BGL3, sh-Bcr-Abl, or EV control. The average volume of tumors induced by control cells is 1. Error bars represent SE. n = 10. * P <
0.05. (d) Schematic representation of the constructs generated for lncRNA-BGL3 overexpression. LncRNA-BGL3 was subcloned into the lentiviral vector pll3.7 to generate pll3.7-lncRNA-BGL3 in which the transcription of lncRNA-BGL3 and GFP was initiated using two independent promoters (EF-1a and CMV respectively). (e)
Lentiviruses encoding lncRNA-BGL3 or the control (pll3.7) were generated in 293T cells. K562 cells were then infected with these viruses, and GFP-positive K562 cells stably expressing lncRNA-BGL3 or pll3.7 were sorted by flow cytometry. Shown are micrographs obtained from fluorescent microscope (Axiovert 200M; Zeiss). (f) Nude mice were subcutaneously injected with K562 cells overexpressing lncRNA-BGL3 or pll3.7. Tumors were excised from mice. Shown are representative images from three independent experiments with similar results (See also Figures 3i-k). (g) Survival of
K562 cells overexpressing lncRNA-BGL3 or pll3.7 was analyzed by flow cytometry after treatment with imatinib. Results are presented as mean ± SEM ( n = 3), * P <
0.05.
Figure S4. Generation of lncRNA-BGL3 expressing transgenic mice. (a) The lncRNA-BGL3 expressing transgenic mice were generated by the microinjection method and genotyped by PCR. RT-PCR analysis was performed to determine the expression efficiency. The transgenic founders with high expression efficiency were
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selected and maintained on a C57BL/6J genetic background. Shown is a representative photograph of lncRNA-BGL3 expressing transgenic mouse and wild-type littermate. (b) Shown is representative genotyping of lncRNA-BGL3 expressing transgenic mice. Numbers 1-16 represent sixteen 14-days old mice. P, positive control; N, negative control.
Figure S5. A miRNA-dependent cross-regulation between lncRNA-BGL3 and
PTEN. (a) Expression of miRNAs shared by lncRNA-BGL3 and PTEN in K562 cells was determined by quantitative real-time PCR analysis. Results are presented as mean
± SEM ( n = 3).
(b) Human peripheral blood mononuclear cells were fractionated into nuclear and cytoplasmic fractions, followed by RNA extraction from each of these fractions and un-fractionated cells. RT-PCR was performed to examine the levels of lncRNA-BGL3, PTEN, nuclear control transcript (U6), and cytoplasmic control transcript (GAPDH) in each of these fractions.
To compare the levels of PTEN with that of lncRNA-BGL3, RT-PCR was performed using the same amount of cDNA template and primers, PCR cycles, and a similar size of amplicon.
(c) Cytoplasmic
PTEN and lncRNA-BGL3 levels in (b) were quantitated by densitometry. The average value of PTEN levels is 1. Plotted are the average relative levels from three independent experiments. The error bars represent the SEM ( n = 3).
(d-g)
LncRNA-BGL3 expression in K562 cells stably expressing PTEN 3’UTR (d and e),
PTEN-specific shRNA (sh-PTEN) (f and g), or controls was examined by RT-PCR analysis. Shown are representative data from three independent experiments with
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similar results. (e and g), lncRNA-BGL3 levels in (d) and (f) were quantitated by densitometry, and normalized to actin levels. The average value in controls (EV or sh-luc) is 1. Plotted are the average levels from three independent experiments. The error bars represent the SEM ( n = 3), * P < 0.05. (h and i) PTEN mRNA in K562 cells overexpressing lncRNA-BGL3 wild-type (lncRNA-BGL3-wt) (h), lncRNA-BGL3 mutant devoid of specific miRNAs binding sites (lncRNA-BGL3-mu) (i), or empty vector (EV) was analyzed by RT-PCR analysis. (j) RT-PCR analysis of PTEN transcript in lncRNA-BGL3 knockdown or control K562 cells.
Figure S6. Functional involvement of the miRNA recognition sequences of lncRNA-BGL3 in leukemic cell survival. (a) Quantitative real-time PCR analysis of lncRNA-BGL3 expression in K562 cells expressing lncRNA-BGL3 wild type
(lncRNA-BGL3-wt), its mutant devoid of the miRNAs binding sites
(lncRNA-BGL3-mu), or empty vector (EV). Results are presented as mean ± SEM ( n
= 3). (b and c) Truncation constructs of lncRNA-BGL3. The prediction of lncRNA-BGL3 secondary structure was performed by RNAfold analysis (b). The regions highlighted by the arrows and numbers indicate the truncation sites in the lncRNA-BGL3 sequence (c). (d) RT-PCR was performed to examine lncRNA-BGL3 levels in K562 cells overexpressing lncRNA-BGL3 or its mutants (mutant A and mutant B). (e) Cell viability of K562 cells overexpressing lncRNA-BGL3, its mutants
(mutant A and mutant B), or EV was assayed by flow cytometry after treatment with or without imatinib. Results are presented as mean ± SEM ( n = 3), * P < 0.05. (f)
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RT-PCR analysis of lncRNA-BGL3 levels in K562 cells overexpressing lncRNA-BGL3 or its mutant (mutant C).
(g) Cell viability of K562 cells overexpressing lncRNA-BGL3, its mutant (mutant C), or EV was assayed as described in (e). (h) Western blotting was performed to examine the protein levels as indicated. (i) Quantitative real-time PCR analysis of PTEN expression in K562 cells expressing lncRNA-BGL3 alone, lncRNA-BGL3 and PTEN shRNA, or EV. Results are presented as mean ± SEM ( n = 3), * P < 0.05.
Figure S7. Bcr-Abl represses lncRNA-BGL3 expression through c-Myc-dependent DNA methylation. (a) Quantitative real-time PCR analysis of expression of lncRNA-BGL3 in human cancer cells; 7402, HepG2 and Jurkat treated with control medium (mock) or 5-Aza-dc (10

M, 72 h). Results are presented as mean ± SEM ( n = 3), * P < 0.05. (b and c) The expression of c-Myc and lncRNA-BGL3 in K562 cells expressing Bcr-Abl shRNA (b), or treated with imatinib
(c) was detected by RT-PCR analysis.
(d) The levels of lncRNA-BGL3, DNMT3B, and c-Myc in c-Myc knockdown K562 cells were examined using RT-PCR and
Western blotting. (e) The expression of lncRNA-BGL3, DNMT3B, and c-Myc in c-Myc overexpressing K562 cells treated with imatinib alone, or imatinib and
5-Aza-dc, was examined by RT-PCR and Western blot analysis.
Figure S8. Expression of CNOT6L, VAPA, and CDKN1A is regulated by lncRNA-BGL3 in K562 leukemic cells. (a and b) Quantitative real-time PCR
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analysis of expression of VAPA and CNOT6L in K562 cells expressing lncRNA-BGL3 (a), lncRNA-BGL3 shRNA (b), or controls. Results are presented as mean ± SEM ( n = 3). (c) RT-PCR was performed to examine CDKN1A levels in
K562 cells expressing lncRNA-BGL3, lncRNA-BGL3 shRNA, or controls.
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