Fig 2: Exploration of role of METTL3/14 in SKOV3 cells. A The protein expression of METTL3 and METTL14 in SKOV3 cells after EZMLD treatment. B Induction of METTL3/14 cDNA in SKOV3 cells suppressed cell viability as determined by CCK8. C, D Flow cytometry analysis of apoptotic cells and quantification. E Western blot analysis of METTL3/14 and apoptosis-associated proteins. F Change in KTR8 mRNA m6A levels after induction of METTL3/14 cDNA in SKOV3 cells. Data are means of three independent experiments ± SD. ▴▴Compared to the control group, P < 0.01

Fig 3: METTL3 and METTL14-mediated m6A modification upregulated SREBF2-AS1 expression via increasing SREBF2-AS1 transcript stability. (A) The correlation between expression level and m6A modification level of SREBF2-AS1 in 80 HCC tissues. r = 0.7523, p < 0.0001 by Spearman correlation analysis. (B) The correlation between SREBF2-AS1 and METTL3 expression level in 371 HCC tissues, derived from TCGA LIHC dataset. r = 0.3779, p < 0.0001 by Spearman correlation analysis. (C) The correlation between SREBF2-AS1 and METTL14 expression level in 371 HCC tissues, derived from TCGA LIHC dataset. r = 0.2472, p < 0.0001 by Spearman correlation analysis. (D) m6A modification level of SREBF2-AS1 in HuH-7 cells with METTL3 or METTL14 overexpression was measured by MeRIP assays. (E) SREBF2-AS1 expression in HuH-7 cells with METTL3 or METTL14 overexpression was measured by RT-qPCR. (F) m6A modification level of SREBF2-AS1 in HuH-7 cells with METTL3 or METTL14 depletion was measured by MeRIP assays. (G) SREBF2-AS1 expression in HuH-7 cells with METTL3 or METTL14 depletion was measured by RT-qPCR. (H,I) SREBF2-AS1 transcript stability in HuH-7 cells with METTL3 or METTL14 overexpression (H) or depletion (I) over time was measured after blocking new RNA synthesis with α‑amanitin (50 µM). For (D–I), results are shown as mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01 by one-way ANOVA followed by Dunnett's multiple comparisons test.

Fig 4: m6A-modified SREBF2-AS1 induced DNA demethylation at SREBF2 promoter through recruiting FXR1 and TET1. (A) The binding of SREBF2-AS1 to SREBF2 promoter, gene body, or 3’UTR in HuH-7 cells was measured using ChIRP assays with SREBF2-AS1 antisense probes or control probes. GAPDH promoter was employed as negative control. (B) ChIRP assays with SREBF2-AS1 antisense probes were performed in HuH-7 cells with overexpression of wild-type or m6A modification sites mutated SREBF2-AS1 to measure the binding of SREBF2-AS1 to SREBF2 promoter. (C) Schematic structure of SREBF2-AS1 and SREBF2 genomic locus. (D) The binding of SREBF2-AS1 to FXR1 in HuH-7 cells was measured using RIP assays with FXR1 specific antibody. (E) RIP assays with FXR1 specific antibody were performed in HuH-7 cells with METTL3 or METTL14 overexpression to measure the binding of SREBF2-AS1 to FXR1. (F) RIP assays with FXR1 specific antibody were performed in HuH-7 cells with wild-type or m6A modification sites mutated SREBF2-AS1 overexpression to measure the binding of SREBF2-AS1 to FXR1. (G) CUT&RUN assays with FXR1 or TET1 specific antibodies were performed in HuH-7 cells with wild-type or m6A modification sites mutated SREBF2-AS1 overexpression to measure the binding of FXR1 and TET1 to SREBF2 promoter. (H) CUT&RUN assays with FXR1 or TET1 specific antibodies were performed in HuH-7 cells with METTL3 or METTL14 overexpression to measure the binding of FXR1 and TET1 to SREBF2 promoter. (I) CUT&RUN assays with FXR1 or TET1 specific antibodies were performed in HuH-7 cells with SREBF2-AS1 depletion to measure the binding of FXR1 and TET1 to SREBF2 promoter. (J) Bisulfate DNA sequencing of CpG157 from HuH-7 cells with wild type or m6A modification sites mutated SREBF2-AS1 overexpression. (K) Bisulfate DNA sequencing of CpG157 from HuH-7 cells with SREBF2-AS1 depletion. (L) SREBF2 expression was measured by RT-qPCR in HuH-7 cells with SREBF2-AS1 overexpression and concurrent FXR1 depletion. (M) SREBF2 expression was measured by RT-qPCR in HuH-7 cells with SREBF2-AS1 overexpression and concurrent TET1 depletion. (N) The correlation between SREBF2 and FXR1 expression level in 371 HCC tissues, derived from TCGA LIHC dataset. r = 0.3412, p < 0.0001 by Spearman correlation analysis. (O) The correlation between SREBF2 and TET1 expression level in 371 HCC tissues, derived from TCGA LIHC dataset. r = 0.3415, p < 0.0001 by Spearman correlation analysis. For (A,B), and (D–M), results are shown as mean ± SD of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, by one-way ANOVA followed by Dunnett's multiple comparisons test (A,B,E,G–K) or Student’s t test (D,F,L,M).

Fig 5: Elevated JMJD6 acetylation predicts a favorable survival in NSCLC patients. (A) The acetylation level of JMJD6 was detected by immunohistochemistry in 68 cases of lung cancer and precancerous lesions. Representative images are shown on the right and statistical data analysis on the left. The scale bars represent 100 μm. (B) Sixty-eight lung cancer patients were clinically selected and divided into two groups, low (0–7) and high (8–12), according to the expression level of JMJD6-acK375. Then the expression of JMJD6-acK375 in lung cancer was tested whether it was related to gender, age, differentiation degree, tumour size and TNM stage, respectively. (C) The expression of JMJD6-acK375 was examined in 68 lung cancer tissues with TNM stage I-II and stage III-IV. (D) JMJD6-acK375 expression was detected in 68 cases of tumor tissues with tumor size greater than or equal to 5 cm and less than 5 cm. (E) Kaplan-Meier analysis of JMJD6 hyperacetylation level, log-rank test, P = 0.022. (F) The working model elucidates the molecular mechanism through which JMJD6 influences the expression of its downstream target METTL14 both pre- and post-acetylation, consequently impacting the expression of the ferroptosis-related protein SLC3A2. In this process, we demonstrated that acetylated JMJD6 enhances METTL14 expression by elevating H4R3me2a levels, whereas METTL14 overexpression suppresses SLC3A2 expression and impedes the progression of lung cancer cells by facilitating ferroptosis