Fig 1: Metabolic stress induces the expression of ADM2 and the activation of ISR signalling pathways in the thyroid cancer cell line. (A) ADM2 expression in thyroid cell lines. (B) RT‐qPCR analysis for ADM2 mRNA in thyroid cancer cell lines in the presence or absence of rotenone (1 μm) for 24 h (n = 3). (C) RT‐qPCR analysis for ISR and mitokine genes of thyroid cancer cell lines in the presence or absence of rotenone (1 μm) (n = 3). (D) Immunoblot analysis of ADM2 and ISR‐related proteins of thyroid cancer cell line in the presence or absence of rotenone (1 μm). (E) Cell viability test using a wst‐8 assay of a thyroid cancer cell line after 24 h treatment of palmitic acid (0, 25, 50, 100 μm) (n = 5). (F) Oxygen consumption rates (OCR) of thyroid cancer cell lines after 24 h treatment with palmitic acid (0, 25, and 50 μm) (8505C, n = 4; BCPAP, n = 5). CCCP: carbonyl cyanide m‐chlorophenyl hydrazone. (G) RT‐qPCR analysis for ADM2 mRNA in thyroid cancer cell lines treated with 50 μm palmitic acid for 24 h (n = 3). (H) RT‐qPCR analysis for ISR and mitokine genes in a thyroid cancer cell line treated with 50 μm palmitic acid for 24 h (n = 3). (I) Immunoblot analysis for ADM2 and ISR‐related proteins from thyroid cancer cell lines treated with 50 μm palmitic acid for 24 h. Mean ± SEM. *p < 0.05 or **p < 0.01 or ***p < 0.001 (two‐tailed Student's t‐test).
Fig 2: ADM2 is related to the ISR and poor clinicopathological features in human thyroid. (A and B) Correlation analysis of ADM2 with ISR genes (A) GTEx‐Thyroid gland. (B) TCGA‐THCA. (C–E) Comparison of groups divided using ADM2 expression on quartile in TCGA‐THCA. (C) The violin and volcano plots. (D) Overall survival and disease‐specific survival. (E) Bar plots representing the gene set enrichment analysis of Gene‐Ontology biological process. (F) Comparisons in CNUH cohort and (G) Immunoblot analysis for ADM2 in the thyroid gland of PTC patients. Serum level of ADM2 in PTC patients divided by clinicopathological features. Mean ± SEM. *p < 0.05 or **p < 0.01.
Fig 3: Transcriptome analyses revealed that ADM2 is upregulated by metabolic stress in the thyroid gland of LSL‐Braf V600E; TgCreERT2 mice. (A) Volcano plot and heat map of RNAseq analysis from LSL‐Braf V600E; TgCreERT2 mice fed CD or HFD (LSL‐Braf V600E ;TgCreERT2 mice fed CD, n = 2; SL‐Braf V600E ;TgCreERT2 mice fed HFD, n = 3). (B) RT‐qPCR analysis of Adm2 mRNA expression in thyroid gland from LSL‐Braf V600E ;TgCreERT2 mice fed CD or HFD (n = 5). (C) Immunoblot analysis of ADM2, ATF4, DDIT3, and β‐actin of the thyroid gland from LSL‐Braf V600E ;TgCreERT2 mice fed CD or HFD (n = 3). (D) Representative immunohistochemistry images of ADM2 expression in thyroid gland from LSL‐Braf V600E; TgCreERT2 mice fed CD or HFD; scale bars: 50 μm (n = 4). (E) Electron microscopy analysis of thyrocytes from LSL‐Braf V600E; TgCreERT2 mice, black arrows indicate mitochondria; scale bars, 500 nm (n = 5). Mean ± SEM. *p < 0.05 or **p < 0.01 or ***p < 0.001 (two‐tailed Student's t‐test).
Fig 4: ADM2 deficiency slows cell proliferation and migration of thyroid cancer cell lines through ERK signalling. (A) Immunoblot analysis of recombinant AMD2‐induced PKA and ERK signal (n = 3). (B) Proliferation analysis in the thyroid cancer cell line transfected with shADM2 (n = 3). (C) Migration analysis in the thyroid cancer cell line transfected with shADM2 (n = 3). (D) Immunoblot analysis for ADM2, AKT, ERK, STAT3, and NF‐kB in the thyroid cancer cell lines transfected with shADM2. Mean ± SEM. *p < 0.05 or **p < 0.01 or ***p < 0.001 (two‐tailed Student's t‐test).
Supplier Page from OriGene Technologies for ADM2 Human shRNA Lentiviral Particle (Locus ID 79924)