Additional file 3 of FTO suppresses glycolysis and growth of papillary thyroid cancer via decreasing stability of APOE mRNA in an N6-methyladenosine-dependent manner
Creators: name: Huang, Jiapeng
name: Sun, Wei
name: Wang, Zhihong
name: Lv, Chengzhou
name: Zhang, Ting
name: Zhang, Dalin
name: Dong, Wenwu
name: Shao, Liang
name: He, Liang
name: Ji, Xiaoyu
name: Zhang, Ping
name: Zhang, Hao
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Sociology
subject: FOS: Sociology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Inorganic Chemistry
subject: FOS: Chemical sciences

description: Additional file 3: Figure S1. FTO inhibits cell proliferation in PTC. a. Overexpression efficiency of FTO in K1 cells determined by qRT-PCR and western blotting. b. Knockdown efficiency of si-FTO-1/2 in TPC1 cells as determined by qRT-PCR and western blotting. c. Total m6A modification levels after FTO knockdown or overexpression in PTC cells as determined by m6A RNA Methylation Quantification Kit. d-e. CCK-8 assay (d) and colony formation assay (e) showing proliferation ability in K1 cells after FTO overexpression. f-g. CCK-8 assay (f) and colony formation assay (g) showing proliferation ability after FTO knockdown in TPC1 cells. h. Flow cytometry was performed to measure cell cycle distribution after FTO knockdown in K1 cells. i. Flow cytometry was performed to measure cell cycle distribution after FTO overexpression in TPC1 cells. j. Knockdown efficiency of sh-FTO in K1 cells determined by western blotting. *P &lt; 0.05, **P &lt; 0.01. Figure S2. FTO inhibition of tumor growth by modulating glycolytic metabolism in PTC. a. ECAR as determined by Seahorse metabolic analysis after transfection with si-NC and si-FTO in TPC1 cells. b-c. Oxygen consumption rate (OCR) as determined by Seahorse metabolic analysis after transfection with si-NC and si-FTO in PTC cells. d-e. Glucose uptake (d) and Lactate production (e) were determined after transfection with si-NC and si-FTO in TPC1 cells. f. Protein expression of GLUT1, HK-II and LDHA were determined by western blotting after FTO knockdown in TPC1 cells. g-h. CCK-8 assay (g) and colony formation assay (h) showing proliferation ability after transfection with si-NC or si-FTO and simultaneous treatment with or without 2-DG in TPC1 cells. i. ECAR as determined by Seahorse metabolic analysis after FTO overexpression in K1 cells. j-k. Glucose uptake (j) and Lactate production (k) were determined after FTO overexpression in K1 cells. l-m. OCR as determined by Seahorse metabolic analysis after FTO overexpression in PTC cells. n. GLUT1, HK-II and LDHA protein expression as determined by western blotting after FTO overexpression in K1 cells. *P &lt; 0.05, **P &lt; 0.01. Figure S3. APOE mRNA is regulated by FTO-mediated m6A modification which is modulated by m6A reader IGF2BP2. a. Gene ontology (GO) analysis of differentially expressed gene sets in FTO knockdown cells. b. Pathway analysis of elevated m6A peak-related gene sets in FTO knockdown cells. c-f. APOE mRNA and protein expression levels as determined by qRT-PCR and western blotting after FTO overexpression or knockdown in PTC cells. g. Relative APOE mRNA expression level as determined by qRT-PCR after transfection with si-NC or si-FTO and simultaneous treatment with or without DAA in TPC1 cells. h-i. Relative luciferase activity of APOE-Wt and APOE-Mut after FTO overexpression or knockdown in PTC cells. j. Half-life (t1/2) of APOE mRNA as determined by qRT-PCR after FTO knockdown in TPC1 cells. k. Relative expression level of APOE mRNA as determined by qRT-PCR after IGF2BP1–3 knockdown in TPC1 cells. l. Half-life (t1/2) of APOE mRNA after IGF2BP2 knockdown as determined by qRT-PCR in TPC1 cells. Figure S4. APOE is negatively regulated by FTO and modulates glycolysis to regulate PTC proliferation. a-d. Knockdown efficiency of si-APOE-1/2 as determined by qRT-PCR and western blotting in PTC cells. e-f. CCK-8 assay (e) and colony formation assay (f) showing proliferation ability after transfection with si-FTO or co-transfection with si-FTO and si-APOE in TPC1 cells. g. ECAR as determined by Seahorse metabolic analysis after transfection with si-FTO or co-transfection with si-FTO and si-APOE in TPC1 cells. h-i. OCR as determined by Seahorse metabolic analysis after transfection with si-FTO or co-transfection with si-FTO and si-APOE in PTC cells. j-k. Glucose uptake (j) and Lactate production (k) after transfection with si-FTO or co-transfection with si-FTO and si-APOE in TPC1 cells. l. Protein expression level of GLUT1, HK-II and LDHA as determined by western blotting after APOE knockdown in TPC1 cells. m. CCK-8 assay showing proliferation ability after transfection with empty vector or APOE and simultaneous treatment with or without 2-Deoxyglucose (2-DG) in TPC1 cells. n-o. Colony formation assay showing proliferation ability after transfection with empty vector or APOE and simultaneous treatment with or without 2-Deoxyglucose (2-DG) in PTC cells. Figure S5. FTO and APOE regulate glycolysis of PTC by modulating IL-6/JAK2/STAT3 signaling pathway. a. Differential gene profiles based on GSEA of APOE gene expression profile data of TCGA. b. Expression level of IL-6/JAK2/STAT3 signaling pathway related proteins as determined by western blotting after FTO knockdown in TPC1 cells. c. Expression levels of IL-6/JAK2/STAT3 signaling pathway related proteins as determined by western blotting after FTO overexpression in K1 cells. d. Expression level of IL-6/JAK2/STAT3 signaling pathway related proteins as determined by western blotting after APOE knockdown in TPC1 cells. e. Expression levels of IL-6/JAK2/STAT3 signaling pathway related proteins as determined by western blotting after APOE overexpression in K1 cells. f. Expression level of GLUT1 protein and IL-6/JAK2/STAT3 signaling pathway related proteins as determined by western blotting after co-transfection with si-FTO and si-APOE in TPC1 cells.