Additional file 2 of The circular RNA circHMGB2 drives immunosuppression and anti-PD-1 resistance in lung adenocarcinomas and squamous cell carcinomas via the miR-181a-5p/CARM1 axis
Creators: name: Zhang, Ling-Xian
name: Gao, Jian
name: Long, Xiang
name: Zhang, Peng-Fei
name: Yang, Xin
name: Zhu, Shu-Qiang
name: Pei, Xu
name: Qiu, Bai-Quan
name: Chen, Shi-Wei
name: Lu, Feng
name: Lin, Kun
name: Xu, Jian Jun
name: Wu, Yong-Bing
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Biological Sciences not elsewhere classified
subject: Cancer
subject: Hematology
subject: Virology

description: Additional file 2: Supplementary Fig. 1. A. The expression of circUSP7 was measured in 3 pairs of NSCLC tissues and matched normal tissues. CircUSP7 was used as a positive control in this study. B. The expression of circHMGB2 was analyzed according to tumor diameter (&lt; 2 cm vs. ≥ 2 cm), lymph node metastasis status (yes vs. no), and TNM stage in 78 LUAD patients. C, Survival analysis of the recurrence and OS of 78 LUAD patients divided into groups according to circHMGB2 expression (circHMGB2high vs. circHMBG2low) was performed using Kaplan–Meier and log rank analysis. Data are presented as the means ± SD; n = 3, *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.0001. Supplementary Fig. 2. A. The expression of circHMGB2 was measured in HBE, LLC and 5 NSCLC cell lines (NCI-H460, A549, PC9, H1703, and NCI-H1299 cells) using qRT–PCR. B. The transfection efficiency of three stable cell lines, A549-circHMGB2, H1299-shcircHMGB2 and LLC-circHMGB2, was validated by qRT–PCR. C. The invasion of A549-circHMGB2 and H1299-shcircHMGB2 cells was assessed by Matrigel Transwell assay. D. The migration of A549-circHMGB2 and H1299-shcircHMGB2 cells was assessed by wound healing assay. E, IHC staining of CD4+ T cells, Tregs, and TAM in subcutaneous tumors derived from LLC-control and LLC-circHMGB2 cells. Data are presented as the means ± SD; n = 3, ****P &lt; 0.0001, ns: not significant. Supplementary Fig. 3. A. Electron microscopy image of exosomes in the supernatants of H460 cells. B. The levels of biomarkers of exosomes from the supernatants of NSCLC cells were measured by western blotting. C. The expression of circHMGB2 in exosomes derived from the supernatants of H460-control and H460-circHMGB2 cells was measured by qRT–PCR. Data are presented as the means ± SD; n = 3, ns: not significant. Supplementary Fig. 4. A. The RNA pulldown assay was performed with PC9, A549 and H1299 cells transfected with biotinylated miR-181a-5p. B, The luciferase activity of SIRT1 was measured in HEK-293 T cells transfected with miR-181a-5p. C. The luciferase activity of CDK8 was measured in HEK-293 T cells transfected with miR-181a-5p. D. The luciferase activity of IRS2 was measured in HEK-293 T cells transfected with miR-181a-5p. E. The luciferase activity of BHLHE40 was measured in HEK-293 T cells transfected with miR-181a-5p. Data are presented as the means ± SD; n = 3, *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ***P &lt; 0.0001, ns: not significant. Supplementary Fig. 5. A. Comparison of the nucleotide sequences between hsa-miR-181a-5p and mmu-miR-181a-5p. B. The putative binding sites of murine Carm1 and miR-181a-5p. C. The luciferase activity of Carm1 was measured in HEK-293 T cells transfected with miR-181-5p. D. The expression of Carm1 was measured in LLC cells overexpressing both circHMGB2 and miR-181a-5p using qRT–PCR. E. The expression of Carm1 was measured in LLC cells overexpressing both circHMGB2 and miR-181a-5p using western blotting. Data are presented as the means ± SD; n = 3, ****P &lt; 0.0001, ns: not significant. Supplementary Fig. 6. A, MS (mass spectrometry) was performed in PC9, A549 and H1299 cells respectively. B, The Co-IP assay was performed in A549 cells to confirm the interaction between CARM1 and HDAC3. C, The expression of type 1 IFN response-related genes was measured in circHMGB2-overexpressing or CARM1-knockout LLC cells after stimulation with IFN-γ for 48 h. Data are presented as the means ± SD; n = 3, ***P &lt; 0.001, ***P &lt; 0.0001, ns: not significant.