Additional file 1 of Long non-coding RNA NEAT1 promotes bone metastasis of prostate cancer through N6-methyladenosine
Creators: name: Wen, Simeng
name: Wei, Yulei
name: Zen, Chong
name: Xiong, Wei
name: Niu, Yuanjie
name: Zhao, Yu
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Supplemental Fig. 1. m6A of NEAT1–1 was elevated in prostate cancer and was a negative prognostic factor for patients. (a) Box and whisker plot showing NEAT1–1 RNA signals upregulated in prostate cancer tissues compwered to normal tissues in TCGA data set. Data from GEPIA. (b) Box and whisker plot showing NEAT1 m6A signals upregulated in bone metastatic prostate cancer tissues. Analyswas of Tianjin Medical University data sets with fresh samples for levels of m6A and NEAT1 RNA were based on the m6A-RIP and RT-PCR. n = 30 each group. P values were shown in the figures. (c) Kaplan-Meier survival analyswas of the TCGA data set for the relationship between the levels of NEAT, expression of NEAT1–1 and survival time in prostate cancers. Data from GEPIA. Supplemental Fig. 2. m6A sites and secondary structure in NEAT1. (a) m6A RIP-seq analyswas of m6A sites of FRMD8 by two independent antibodies. The m6A profiles of FRMD8 were shown in genome browser. (b) NEAT1–1 and NEAT1–2 expressions were measured by qRT-PCR in P-18 primary cells. M6A levels of NEAT1–1 and NEAT1–2 were measured by m6A-RIP-PCR in P-18 primary cells. Means and standard deviations (error bar) were determined from three replicates. Error bars represent mean ± SD for triplicate experiments. P values were shown in the figures. (c-f) Secondary structure of NEAT1–1 predicted by https://rna.tbi.univie.ac.at . m6A putative motif and sequences were shown in each figures. (g) NEAT1–1 expressions were measured by qRT-PCR in 293 T and P-18 primary cells. Transfected NEAT1–1 expressions were measured by qRT-PCR using primers targeting NEAT1–1 and plasmid in P-18 primary cells. Means and standard deviations (error bar) were determined from three replicates. Error bars represent mean ± SD for triplicate experiments. P values were shown in the figures. Supplemental Fig. 3. CYCLINL1 and CDK19 in tissues. (a) Expression of METTL3 and Vinculin proteins were measured by western blot in P-18 METTL3-KO cells infected with control or METTL3 KO CRWASPR-Cas9 plasmids. (b) Amino acid sequence comparwason of CDK8 and CDK19. (c) Dot blot of m6A and biotin to testing NEAT1–1 WT and m6A-deletion probes using anti-m6A and anti-biotin antibodies. 0.5 μg RNA for each dot. (d) CYCLINL1 and CDK19 RNA levels in normal human tissues. Data from GTEx. (e and f) Box and whisker plot showing CYCLINL1 and CDK19 signals upregulated in bone metastatic prostate cancer tissues. Analyswas of Tianjin Medical University data sets for levels of CYCLINL1 and CDK19 RNA were based on the RT-PCR. n = 30 each group. Supplemental Fig. 4. NEAT1–1 interacted with CYCLINL1 and CDK19. (a) Left panel, dot blot of m6A to testing NEAT1–1 full length RNA using anti-m6A. 0.5 μg RNA for each dot. Methylene blue was used to measure input loading. Right panel, 1 μg GST or GST-Pol II-C terminal fusion proteins and 2 μg NEAT1–1 RNA were incubated with CDK19 and CYCLINL1 produced from Quick coupled transcription/translation kit. After extensive washing, GST-beads were collected and subject to SDS/PAGE and western blot with Pol II Ser-2p, total Pol II and GST antibodies. (b) GST or GST-Pol II-C terminal fusion proteins and 2 μg NEAT1–1 RNA were incubated with CDK19 and CYCLINL1 WT or mutated proteins produced from Quick coupled transcription/translation kit from T7 promoter. After extensive washing, GST-beads were collected and subject to SDS/PAGE and western blot with Flag, Myc, Pol II Ser-2p, total Pol II and GST antibodies. (c) NEAT1–1 RNA (green) and AR RNA (red) were stained using probes with FITC or TEXAS-RED dye. Scale bar was 10 μm. (d) NEAT1–1, MALAT1, Xist and ARLNC1 expressions were measured by qRT-PCR in P-18 primary cells. Means and standard deviations (error bar) were determined from three replicates. Error bars represent mean ± SD for triplicate experiments. P values were shown in the figures. (e) NEAT1–1 site#4 m6A levels were measured by m6A RIP PCR using primers targeting site#4 in P-18 primary cells. Means and standard deviations (error bar) were determined from three replicates. Error bars represent mean ± SD for triplicate experiments. P values were shown in the figures. Supplemental Fig. 5. Sanger sequence showed NEAT1–1 knocking out in P-18 cells. (a) gRNAs sequences were shown in the two sides of NEAT1–1 RNA in genome. (b) NEAT1–1 full length and knocking out PCR products were amplified using primers outside the NEAT1–1. (c) gRNAs sequences were shown in the possible target in chromatin 22. (d) Target genes’ expressions were measured by qRT-PCR in P-18 NEAT1–1 KO cells transfected with NEAT1–1 WT and NEAT1–1 m6A-mut. Means and standard deviations (error bar) were determined from three replicates. Error bars represent mean ± SD for triplicate experiments. P values were shown in the figures. Supplemental Fig. 6. X-ray for detection of mice bone. (a) Mice were scanned using a microcomputed x-ray system. Representative images from 10 different samples were shown. (b) NEAT1–1 expressions were measured by qRT-PCR in P-18 and P-34 xenografts. Means and standard deviations (error bar) were determined from six replicates (n = 6). Error bars represent mean ± SD for triplicate experiments. NS, no significance.