MOESM3 of A novel IFNα-induced long noncoding RNA negatively regulates immunosuppression by interrupting H3K27 acetylation in head and neck squamous cell carcinoma
Creators: name: Hailong Ma
name: Hanyue Chang
name: Wenyi Yang
name: Yusheng Lu
name: Jingzhou Hu
name: Shufang Jin
Publisher: name: figshare
Year: 2020
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 3: Figure S1. The IFNα concentration in medium supernatant from HNSCC cells, normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs) was measured via ELISA. The IFNα concentration was also detected after CDDP (2.5 μg/ml), 5-FU (10 μg/ml), cetuximab (200 ng/ml) and erlotinib (1.5 μM) treatment for 24 h in HNSCC cells. Figure S2. Differentially expressed lncRNAs were detected via sequencing after treatment with 200 ng/ml IFNα for 24 h. Differentially expressed lncRNAs were detected via sequencing after treatment with 200 ng/ml IFNα for 24 h. Figure S3. The nucleotide sequence of lncMX1-215 was identified using RACE (RACE for EGFR served as the positive control). Figure S4. LncMX1-215 chromatin location and encoding structure determined by RACE analysis was shown. Figure S5. LncMX1-215 localization was analyzed in Cal27 cells using PCR. U6 RNA and β-actin were used as the positive controls for nuclear RNA and cytoplasmic RNA, respectively. Figure S6. LncMX1-215 expression was detected in tumor and adjacent normal tissues from HNSCC patients. Figure S7. LncMX1-215 expression was analyzed after treatment with 200 ng/ml IFNα and 0.5 μM fludarabine for 24 h. Figure S8. LncMX1-215 expression was quantified using RT-PCR after stat1-specific siRNA transfection and then treatment with 200 ng/ml IFNα for 24 h. Figure S9. ChIP assays were performed using isotype IgG antibody after treatment with 200 ng/ml IFNα for 24 h. Figure S10. ChIP assays were conducted to analyze lncMX1-215 promoter binding under 200 ng/ml IFNα and 0.5 μM fludarabine treatment for 24 h. Figure S11. Cells were pretreated with 100 ng/ml rhGalectin-9 and then incubated with NK cells for 4 h. The specific lysis rate was measured using an LDH kit. Figure S12. a PD-L1 and acetylation of histone 3 were detected and quantified after treatment with 200 ng/ml IFNα or 15 μM SAHA for 24 h. b PD-L1, H3K27ac and H3K9ac were detected and quantified after the indicated SAHA treatment for 24 h. c PD-L1 and H3K27ac were detected and quantified after 15 μM SAHA treatment for the indicated time.# indicated the difference between combined group and each alone. *P &lt; 0.05, and **P &lt; 0.01. Figure S13. Galentin-9 expression was detected in HN4 and Cal27 cells after SAHA or MS-275 treatment for 24 h. Figure S14 The promoter activity of PD-L1 and LGALS9 was measured after transfection with lncMX1-215 for 24 h and then 1.5 μM SAHA or 0.5 μM MS-275 treatment for 24 h in 293T cells.Fig. S15. After ectopic expression of GCN5 and vector or lncMX1-215 for 48 h in HN4 and Cal27 cells, ChIP assay was performed to analyze the binding to PD-L1 promoter using anti-GCN5 antibody. Figure S16. GCN5 and H3K27ac expression was detected using immunofluorescence in HN4 and Cal27 cells after lncMX1-215 transfection for 48 h. Figure S17. The expression of H3K27ac and GCN5 was detected using immunofluorescence in HNSCC TMA. Figure S18. RIP assays were performed with HN4 cells. Figure S19. A linear lncMX1-215 template was constructed after restriction enzyme digestion of the pcDNA3.1 recombinant vector. Figure S20. EdU assays were performed after transfection of HN4 and Cal27 cells with the indicated constructs; magnification: ×100. Figure S21. Tumors on the bilateral flank of nude mice were shown. Figure S22. TUNEL assays were conducted to assess the number of apoptotic cells in xenograft tumor sections; magnification: ×200. Figure S23. Ki-67 staining of xenograft tumor sections was performed. Figure S24. LncMX1-215 inhibited tumorigenesis and lung metastasis in SCC7-bearing mice. a SCC7-bearing xenografts were established in C3H mice and the tumors were resected and measured at experimental endpoint (n=5/group). b Lung metastasis assay was performed using SCC7 cells in C3H mice and the metastasis nodules were counted and analyzed (n=3/group).