Fig 2: METTL16 mediated downregulation of TIALD in HCC.A LncRNA pulldown combined with mass spectrometry to Identify TIALD interaction proteins (left) and list of potential interacting proteins of TIALD (right). B RNA pull-down assay was performed in SMMC-7721 and SNU449 cells using biotin-labeled TIALD RNA probe transcribed in vitro, and METTL16 was detected using western blots. C RIP-qPCR analysis of the interaction of METTL16 and TIALD in SMMC-7721 and SUN449 cells. Enrichment of TIALD with METTL16 antibody and IgG control was measured by qPCR. D METTL16 was knock-down or over-expressed in SMMC-7721 and SNU449 cells, the expression of METTL16 was detected by western blots and the expression of TIALD was detected by qPCR. E FISH analysis of the expression of TIALD in METTL16 over-expression SMMC-7721 cells and METTL16 knockdown SNU449 cells. F The correlation between METTL16 and TIALD in TCGA datasets (up) and qRT-PCR analysis of METTL16 and TIALD in 33 HCC tissues (down). G Wild-type and different fragment of TIALD sequences were cloned into a luciferase reporter (up). Relative luciferase activity of the wild-type and different fragment of TIALD reporter vectors catalyzed by METTL16 (down).

Fig 3: m6A modification is involved in the degradation of TIALD.A MeRIP-qPCR analysis was used to demonstrate METTL16-mediated TIALD m6A modification in SMMC-7721 and SNU449 cells. m6A modification of TIALD was significant enriched upon METTL16 over-expression. B The expression levels of TIALD in METTL16 or shMETTL16 cells were quantified by real-time PCR at indicated time points after actinomycin D treatment and the decay rate of TIALD was evaluated with a linear regression model. C ALKBH5 up-regulated the expression of TIALD. ALKBH5 was over-expressed in SMMC-7721 and SNU449 cells, and the expression of TIALD and ALKBH5 were detected using qPCR. D YTHDF2 and YTHDC1 negatively regulated TIALD expression. YTHDF2 and YTHDC1 was knock-down in SMMC-7721 cells and the expressions of TIALD, YTHDF2 and YTHDC1 were detected using qPCR.

Fig 4: METTL16 exerts its anti-ferroptotic effect mediated by SENP3. (A-B) Intracellular lipid peroxidation (A) and ROS (B) content of Hep3B were observed (RSL3:1µM). (C) Intracellular lipid peroxidation and ROS content of Hep3B were quantified (RSL3:1µM). (D) Intracellular GSH levels, Iron content, and MDA levels of Hep3B (RSL3:1µM). (E) Representative images and statistical data of weights and volumes of xenografts from Hep3B (n = 5 mice per group). (F) Representative images of HE staining and METTL16, SENP3, MDA, 4-HNE IHC staining of xenografts from Hep3B. Scale bar, 100 μm. Statistical significance was analyzed using one-way ANOVA with Tukey’s test (C, D and E, n = 3 or 5), and two-way ANOVA with Tukey’s test (E, n = 5)

Fig 5: METTL16/IGF2BP2 facilitates the mRNA stability of SENP3 in an m6A-dependent manner. (A) The top 5 genes with the highest correlation coefficients linked to METTL16. (B) Correlation between METTL16 and SENP3 expression in public HCC databases. (C-D) The levels of METTL16 and SENP3 in the indicated cells. (E) Luciferase activities of the pGL3.0 plasmids containing SENP3 CDS and 3’UTR. (F) Schematic representation of the potential m6A sites and the mutation of m6A sites (T-A), as well as the primers used for MeRIP/RIP qPCR on SENP3 mRNA. (G) Luciferase activities of the pGL3.0 plasmids containing WT or MUT SENP3 CDS/3’UTR. (H) The relative m6A enrichment levels at the indicated site within the SENP3 mRNA via MeRIP-qPCR analysis. (I) RIP-qPCR was used to detect the binding of METTL16 and SENP3 at the indicated site. (J) The decay rate of SENP3 mRNA upon ActD (5 µg/mL) treatment at the indicated time points in PLC/PRF/5. (K-L) The SENP3 level was detected after multiply silencing different m6A readers in the indicated cells. (M) RIP-qPCR was used to detect the binding of IGF2BP2 and SENP3 at the indicated site upon METTL16 overexpression. (N) The decay rate of SENP3 mRNA in PLC/PRF/5. Statistical significance was analyzed using two-tailed Student’s t test (C, G, I, K and M, n = 3), one-way ANOVA with Tukey’s test (C, E and H, n = 3), two-way ANOVA with Tukey’s test (L, n = 3) and Pearson correlation analysis (B)