Journal: Theranostics

Article Title: SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma.

doi: 10.7150/thno.42539

Figure Lengend Snippet: Figure 5. SUMOylation of Mettl3 regulates Snail mRNA homeostasis via m6A methyltransferase. (A) Mettl3-WT or -KR was transiently transfected into MHCC97H cells and detected by the dot-blot assay with the anti-m6A antibody. Equal loading of mRNAs was confirmed by methylene blue staining. (B) Mettl3 with or without Ubc9 was transfected into MHCC97H cells and detected by the dot-blot assay with the anti-m6A antibody. Equal loading of mRNAs was confirmed by methylene blue staining. (C) Suppression or overexpression of Mettl3 in HCC was determined by RT-qPCR, and GAPDH was used as the normalized control. (D) mRNA of Snail in Mettl3 suppression or overexpression cells. (E) Mettl3-WT or Mettl3-KR-expressing cells were transfected with the pEZX-PL01-Snail promoter reporter plasmid and negative control plasmid for 36 h. Results were expressed as the ratios between F-luc and R-luc activities. (F) IP immunoblot analysis was performed in Mettl3-WT- and Mettl3-KR-expressing cells with the anti-Mettl3 antibody, followed by western blotting with Mettl3, anti-Eef2, anti-eIF4E, and anti-NCBP1 antibodies. One-tenth of lysates as the input was immunoblotted with indicated antibodies. (G) Mettl3-WT- and Mettl3-KR-expressing cells were pretreated with MG-132 for 6 h and stimulated with serum for indicated times. Subsequently, Mettl3 and Snail protein expression levels were analyzed by western blotting. (H) Scramble or siMettl3-expressing cells were fed with CHX for the indicated times, and protein expression of Mettl3 and Snail was analyzed by western blotting. (I) Mettl3-WT or Mettl3-KR-expressing cells transfected with different plasmids were treated with CHX for the indicated times, and protein expression of Mettl3 and Snail was detected by western blotting. (J and K) The decay rate of mRNA and qPCR analysis of Snail at the indicated times after exposure to the transcription inhibitor actinomycin D (5 μg/mL) in MHCC97H (J) and HepG2 (K) cells. The relative expression level was normalized to β-actin. Data are presented as mean ± s.d. * p < 0.05, ** p < 0.01; Student’s t-test.

Article Snippet: The primary antibodies, including anti-GAPDH, anti-LaminB1, anti-His, anti-HA, anti-Flag, anti-MMP2 (from proteintech), anti-Mettl3 (from abcam, Bethyl); anti-m6a (from Synaptic Systems); SUMO-1 and SUMO2/3 (from abcam); anti-MMP9, anti-E-cadherin and normal rabbit IgG (from CST); UBC9, Snail and normal mouse IgG (from santa cruz); anti-rabbit, Anti-mouse peroxidase-conjugated secondary antibodies (from proteintech). m6A dot blot assay mRNA was enriched using Dynabeads mRNA DIRECT Purification Kit (InvitrogenTM, 61012).

Techniques: Transfection, Dot Blot, Staining, Over Expression, Quantitative RT-PCR, Control, Expressing, Plasmid Preparation, Negative Control, Western Blot