Additional file 1 of The lncRNA LAMP5-AS1 drives leukemia cell stemness by directly modulating DOT1L methyltransferase activity in MLL leukemia
Creators: name: Wang, Wen-Tao
name: Chen, Tian-Qi
name: Zeng, Zhan-Cheng
name: Pan, Qi
name: Huang, Wei
name: Han, Cai
name: Fang, Ke
name: Sun, Lin-Yu
name: Yang, Qian-Qian
name: Wang, Dan
name: Luo, Xue-Qun
name: Sun, Yu-Meng
name: Chen, Yue-Qin
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1. Figure S1. Identification of LAMP5-AS1 transcripts. a Agarose gel for the 5’ and 3’ RACE identified LAMP5-AS1 transcripts in THP1 cells. Two 5’-ends and one 3’-end of LAMP5-AS1 variant cDNA in cells were identified by the nested PCR. Schematic depiction of the two LAMP5-AS1 transcripts (bottom). b qRT-PCR for the relative expression of the two different LAMP5-AS1 transcripts. Figure S2. Impact of LAMP5-AS1 on primary MLL leukemia differentiation. a LAMP5-AS1 was highly expressed in all MLL leukemia cell lines labeled by * (analyzed by △CT). b qRT-PCR analysis for LAMP5-AS1 knockdown in 4 primary cells from patients with MLL leukemia, including three ALL with MLL-AF9 and MLL-AF4 and an AML with MLL-AF10, respectively, after transduction with LAMP5-AS1 siRNAs or control. Error bars reflect ± SEM (*, p &lt; 0.05, **, p &lt; 0.01) in three independent experiments. c Representative graph for the flow cytometric analysis of the CD19+, CD11b+, or CD14+ cell populations in the primary MLL leukemia cells. d Representative graph for the flow cytometric analysis of the CD34+ cell populations in primary MLL leukemia cells. Histogram plots show the statistical values. Error bars reflect ± SEM (*, p &lt; 0.05, **, p &lt; 0.01) in three independent experiments. Figure S3. LAMP5-AS1 plays a role in MLL leukemia cell maintenance. a, b qRT-PCR analysis for LAMP5-AS1 knockdown in MLL leukemia cells, after transduction with LAMP5-AS1 siRNAs or control (a) and LAMP5-AS1 shRNAs or control (b). Error bars reflect ± SEM (**, p &lt; 0.01; ***, p &lt; 0.001) in three independent experiments. c-e Representative flow cytometry graphs showing the CD14 (c), CD11b (d), and CD19 (e) cell populations in MLL leukemia cells treated with LAMP5-AS1 knockdown relative to those levels in control. The values were analyzed by Error bars reflect ± SEM (*,p &lt; 0.05, **,p &lt; 0.01,***, p &lt; 0.001) in three independent experiments. f Morphology of colonies of MLL leukemia cells 10 days upon shRNA-mediated knockdown of LAMP5-AS1. Scale bars, 100 μm. Error bars reflect ± SEM (***, p &lt; 0.001) in three independent experiments. Figure S4. Identification of LAMP5-AS1 binding to DOT1L in cell nucleus. a We fractionated the nucleus and cytoplasm from the THP1 cells and found that LAMP5-AS1 predominantly localizes to the cell nucleus, with NEAT1 as a nuclear marker and hY1 as a cytoplasmic marker. Error bars reflect ± SEM (***, p &lt; 0.001) in three independent experiments. b RNA FISH showing most of LAMP5-AS1 localizes in the nuclei of MLL leukemia cells. Scale bars, 5 μm. c Agarose gel showing the templates of LAMP5-AS1 and LAMP5-AS1 antisense in the RNA-pull-down assay. d Agarose gel showing the PCR template of DOT1L. e Western blotting of DOT1L-N-FLAG in the products of RIP, with beta-tubulin as the negative control. Cell lysis harvested from the DOT1L-N-FLAG stably expressed THP1 cells. f RIP of DOT1L-FLAG in MOLM13 indicating that LAMP5-AS1 was significantly enriched compared with U6, actin, and GAPDH. g RNA FISH and IF experiments showed that LAMP5-AS1 co-localizes with DOT1L in the nuclei of MV4-11 cells. Scale bars, 5 μm. h Agarose formaldehyde gel showing the in vitro RNA transcription of LAMP5-AS1 sections. Biotin labeled UTP was added in the reaction. Figure S5. Epigenomic changes upon LAMP5-AS1 knockdown. a ChIP-seq profiles of H3K79me2 and H3K79me3 at the CDK6 genomic loci in LAMP5-AS1-knockdown (green) compared with control (gray) MOLM13 cells. The y-axis scales represent read density per million sequenced reads. b H3K79me2(left) and H3K79me3(right) ChIP-qPCR for the core target genes of MLL fusion protein in the LAMP5-AS1 knockdown (red) compared with control (gray) established MOLM13 cells. Error bars reflect ± SEM (*, p &lt; 0.05) from three independent experiments. c Representative meta-analysis plot showing H3K79me2 profile across the +10 kb to -10 kb genomic region around the TSS of MLL-AF9 target genes. Profiles of LAMP5-AS1-knockdown (green) compared with control (blue) MOLM13 cells are presented. Figure S6. Genomic changes upon LAMP5-AS1 knockdown or overexpression. a qRT-PCR analysis determined that the expression levels of the MLL fusion protein target genes including HOXA9, HOXA10 and MEIS1 were decreased upon LAMP5-AS1 knockdown in MV4-11 cells. Error bars reflect ± SEM (*, p &lt; 0.05, **, p &lt; 0.01; ***, p &lt; 0.001) in three independent experiments. b qRT-PCR analysis determined that the expression levels of the MLL fusion protein target genes including HOXA9, HOXA10 and MEIS1 were decreased upon LAMP5-AS1 knockdown in 4 primary MLL leukemia cells. Error bars reflect ± SEM (*, p &lt; 0.05, **, p &lt; 0.01; ***, p &lt; 0.001) in three independent experiments. c Western blotting for the protein levels of HOXA9 and Mesi1 in MLL leukemia cells transduced by LAMP5-AS1 siRNA and control. d Overexpression of LAMP5-AS1 transcript 1 in MLL leukemia cells (MOLM13, MV4-11, and THP1). e qRT-PCR analysis determined that the expression levels of the MLL fusion protein target genes including HOXA9, HOXA10 and MEIS1 were increased in MLL leukemia cell lines treated with LAMP5-AS1 overexpression. Error bars reflect ± SEM (*, p &lt; 0.05, **, p &lt; 0.01; ***, p &lt; 0.001) in three independent experiments. f Immunoblot showing the protein levels of HOXA9 and Mesi1 upregulated upon overexpression of LAMP5-AS1 in MLL leukemia cell lines. Table S1. Patient demographics and clinicopathologic features. Table S2. Demographics and clinicopathologic features of primary MLL leukemia patient samples. Table S3. The primers used in this work. Table S4. siRNA/shRNA. Table S5. ALL of the antibodies and regents used in this study. Table S6. MS of proteins from LAMP5-AS1 pull down.