Additional file 2: of A novel long non-coding RNA from the HOXA6-HOXA5 locus facilitates colon cancer cell growth
Creators: name: Saijo, Saki
name: Kuwano, Yuki
name: Tange, Shoichiro
name: Rokutan, Kazuhito
name: Kensei Nishida
Publisher: name: figshare
Year: 2019
Subjects: subject: Biophysics
subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology

description: Figure S1. Schematic representation of the primer sets used for the qPCR assay and siRNAs targeting of the HOXA6-HOXA5 locus. Primers for qPCR amplification of the indicated transcripts or genomic regions are indicated by the left/right arrows. Transcripts, HOXA5 short and HOXA5 long 1; regions, HOXA5 coding region (CR) and 3′ UTR. Targeted sequences of siRNAs #1 to #8 are indicated by the solid lines and the dashed lines for siRNAs #7 and #8 indicate the skipped sequences of the siRNAs. Figure S2. 5′-rapid amplification of cDNA ends (RACE) experiments of the HOXA6-HOXA5 locus. A. Scheme diagram of the gene-specific primers used for 5′-RACE experiment. B. Electrophoretic analysis of PCR amplification products. C. Nucleotide sequences of the PCR products. Primers used are underlined. Grey boxes indicate the junctions between different exons. M, DNA ladder marker. Figure S3. 3′-rapid amplification of cDNA ends (RACE) experiments of the HOXA6-HOXA5 locus. A. Scheme diagram of the gene-specific primers used for 3′-RACE experiment. B. Electrophoretic analysis of PCR amplification products. C. Nucleotide sequences of the PCR products. Primers used are underlined. Grey boxes indicate the junctions between different exons. M, DNA ladder marker. Figure S4. Analysis of translation potency of the HOXA5 short RNA. A. A T7 promoter-containing DNA fragments encoding full-length HOXA5 RNA, HOXA5 short RNA, or GAPDH were generated by PCR amplification and the resultant PCR products were subjected to in vitro transcription and translation assays, which included the incorporation of fluorescent lysine. The synthesized proteins were analyzed by 15% SDS-PAGE and detected using a fluoro-imaging instrument. B. The translation potency of HOXA5 short RNA was calculated using Coding-Potential Assessment Tool (CPAT) software. Sequences of the coding regions of HOXA5 and GAPDH were used as translatable sequences and that of MALAT1, known as a functional long non-coding RNA, was used as an untranslatable sequence. Figure S5. Evolutionary conserved sequences of a transcriptional start site of the HOXA5 short RNA. Sequence alignment of the upstream sequences of a transcriptional start site (TSS) in HOXA5 short RNA indicates the presence of a consensus TATA box and a TSS in most species. Figure S6. Intrinsic chemoresistance to 5-FU in HOXA5 short RNA expressing HCT116 cells. The cell viability of pEB-HOXA5 short or pEB-mock HCT116 cells was determined by Cell Count Reagent SF after treatment with increasing doses of 5-FU for 48 h. Figure S7. Effects of HOXA5 short RNA on AKT and ERK activation. Protein levels of phosphorylated AKT (Ser473; #9271, Cell Signaling Tech.), total AKT (#9272, Cell Signaling Tech.), phosphorylated ERK1/2 (#9101, Cell Signaling Tech.) and total ERK1/2 (#9102, Cell Signaling Tech.) were measured by western blot analysis. GAPDH levels were used as an endogenous quantitative control. The level of phospho-AKT, phosphor-ERK1/2, AKT or ERK1/2 band relative to that of GAPDH was quantitatively analyzed by densitometry. #: The band corresponding to phospho-AKT was not sufficiently detected for densitometry analyses. (PDF 561 kb)