Additional file 1: Figure S1. of Long noncoding RNA MALAT1 regulates autophagy associated chemoresistance via miR-23b-3p sequestration in gastric cancer
Creators: name: YiRen, Hu
name: YingCong, Yu
name: Sunwu, You
name: Keqin, Li
name: Xiaochun, Tong
name: Senrui, Chen
name: Ende, Chen
name: XiZhou, Lin
name: Yanfan, Chen
Publisher: name: Figshare
Year: 2017
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: 60506 Virology

description: Flow cytometric analysis of Annexin V staining. Figure S2 MALAT1 expression levels vary across different cancer types in the TCGA database. Figure S3 (a) MALAT1 expression was detected in SGC7901/VCR cells by qRT-PCR after transduction of lentiviruses encoding MALAT1 shRNA or a scrambled shRNA. Northern blot analysis of MALAT1 expression in SGC7901/VCR cells after transduction of lentiviruses encoding MALAT1 shRNA or a scrambled shRNA. (b) MALAT1 expression was detected in SGC7901/VCR cells by qRT-PCR after transfection of lentivirus harboring the full-length human MALAT1 sequence or the empty vector. The data are presented as the means ± S.D. of values obtained in 3 independent experiments. *, p &lt; 0.05. Figure S4 MALAT1 promotes autophagy. (a) SGC7901/VCR cells stably transfected with full-length human MALAT1 sequence or the empty vector were subjected to Western blot analysis of LC3-II and p62. (b) Autophagy was evaluated using transmission electron microscopy in SGC7901/VCR stably transfected with full-length human MALAT1 sequence or the empty vector. (c) In BGC823 cells, treatment of CDDP (10 μg/ml) for 24 h induced a significant upregulation of MALAT1 as determined with qRT-PCR analysis. (d) In BGC823 cells, treatment of CDDP (10 μg/ml) for 24 h induced a significant activation of autophagy, while MALAT1 knockdown blunted the autophagic response to cisplatin. (e) SGC7901 cells transfected with full-length human MALAT1 sequence or the empty vector were treated with the cisplatin (5 μg/ml) for 24 h, caspase-3 protein was detected by Western blot. The data are presented as the means ± S.D. of values obtained in 3 independent experiments. *, p &lt; 0.05. (f) SGC7901/VCR cells stably transfected with shRNA-lncRNA-ATB or a control were subjected to Western blot analysis of LC3-II and p62. Figure S5 (a) UCSC Genome Bioinformatics Site ( http://genome.ucsc.edu /) showed high enrichment of H3K27Ac at the promoter of MALAT1. (b) ChIP assays detected the H3K27Ac acetylation at promoter of MALAT1 in gastric cancer tissues. (c) ChIP assays detected the H3K27Ac acetylation at promoter of MALAT1 in gastric cancer cells.*, p &lt; 0.05, **, p&lt; 0.01. (d) The expression of the MALAT1 transcript (mean ± standard deviation) was detected using RT-PCR after cells were stimulated with varying concentrations of the histone deacetylase inhibitor trichostatin A (TSA) for 24 hr. Figure S6 (a) Compared with chemosensitive patients, LC3B and MALAT1 were markedly upregulated in chemoresistant patients using immunohistochemical analysis (for LC3B) and in situ hybridization analysis (for MALAT1). (b) According to data from The KMPlot database (TCGA), high MALAT1 expression resulted in a poorer disease-free survival (DFS, n=153, p=0.049) and overall survival (OS, n=153, p=0,039) in patients who received 5-Fu based adjuvant therapy. The HRs and p values were calculated with log-rank tests. Figure S7 (a) The copy number of MALAT1 or miR-23b-3p detected SGC7901/VCR cells, using RT-PCR and standard curves of known copy numbers of plasmid-derived reference standard. Error bars show standard deviation. (b) Cellular characterization of MALAT1 and miR-23b-3p, the levels of nuclear control transcript (U1), cytoplasmic control transcript (Actin mRNA), and MALAT1 were assessed by qRT-PCR in nuclear and cytoplasmic fractions in SGC7901/VCR cells. Data are presented as a percentage of U1, Actin and MALAT1 levels and total levels for each were taken to be 100%. The data are presented as the means ± S.D. of values obtained in 3 independent experiments. Figure S8 (a) In BGC823 cells, treatment of CDDP (10 μg/ml) for 24 h induced a significant downregulation of miR-23b-3p as determined with qRT-PCR analysis. (b) MALAT1 expression was detected in SGC7901/VCR cells by qRT-PCR after transduction of lentiviruses encoding miR-23b-3p mimic or control mimic. (c) SGC7901/VCR cells were transfected with sh-NC, sh-MALAT1, sh-MALAT1+miRNA-23b-3p inhibitor and miR-23b-3p inhibitor. qRT-PCR was performed 48 h post transfection. ATG12 and HMGB2 were determined with qRT-PCR analysis. Figure S9 (a) Compared with chemosensitive patients, ATG12 was markedly upregulated and miR-23b-3p was downregulated in chemoresistant patients using immunohistochemical analysis (for ATG12) and in situ hybridization analysis (for miR-23b-3p). (b,c) According to data from The KMPlot database (TCGA), low miR-23b-3p and high ATG12 expression resulted in a poorer disease-free survival and overall survival in patients who received 5-Fu based adjuvant therapy. The HRs and p values were calculated with log-rank tests. Table S1 Primer sequence used in this study. (DOCX 2642 kb)