Fig 2: Claspin and Bcl-2 might be the targets of YTHDF3 in celastrol-treated pancreatic cancer cells. A Overexpression of YTHDF3 in celastrol (2.0 µM)-treated AsPC-1 and celastrol (2.5 µM)-treated BxPC-3 cells after transfection with the vector encoding YTHDF3 (OE YTHDF3) or vector control (vector) was confirmed by Western blotting. GAPDH served as loading control. n = 3. B Expression of Claspin and Bcl-2 in YTHDF3 overexpressing (OE YTHDF3) and control cells (vector) after incubation with celastrol (2.0 µM for AsPC-1 and 2.5 µM for BxPC-3 cells) was evaluated by Western blotting. GAPDH served as loading control. n = 3. C, D Representative EdU assay in YTHDF3 overexpressing (OE YTHDF3) and control cells (vector) after incubation with celastrol (2.0 µM for AsPC-1 and 2.5 µM for BxPC-3 cells) was shown. Scale bars = 100 μm. E, F Representative cell cycle analysis in YTHDF3 overexpressing (OE YTHDF3) and control cells (vector) after incubation with celastrol (2.0 µM for AsPC-1 and 2.5 µM for BxPC-3 cells) was shown. G, H Representative cellular apoptotic analysis in YTHDF3 overexpressing (OE YTHDF3) and control cells (vector) after incubation with celastrol (2.0 µM for AsPC-1 and 2.5 µM for BxPC-3 cells) was shown. All data were shown as the means ± SD from three independent experiments, *P < 0.05

Fig 3: Celastrol down-regulated Claspin and Bcl-2 expression in an m6A-YTHDF3 mediated pattern in pancreatic cancer cells. A, B The relative levels of Bcl-2 or Claspin mRNA in celastrol-treated both AsPC-1 (A) and BxPC-3 cells (B) after incubation with actinomycin D (2 μg/ml) for the indicated times (normalized to 0 h) were determined by RT-qPCR. C RIP-qPCR assay was performed to evaluate the interaction between YTHDF3 and the mRNA of Claspin or Bcl-2 in AsPC-1 and BxPC-3 cells in the presence or absence of celastrol. D, E The relative levels of Claspin or Bcl-2 protein in celastrol-treated AsPC-1 and BxPC-3 cells after incubation with cycloheximide for the indicated times were determined by Western blotting (D) and quantitatively analyzed (E). All data were shown as the means ± SD from three independent experiments, *P < 0.05

Fig 4: FTO decreases TLR4 expression by mediating the m6A modification of TLR4. (A) TLR4 expression in cardiomyocytes after overexpression of FTO tested with RT-qPCR and western blotting. (B) The m6A modification level of TLR4 mRNA examined with the Me-RIP assay. (C) RIP assay to determine TLR4 expression after overexpression of FTO. (D) TLR4 expression after knockdown of YTHDF1, YTHDF2, and YTHDF3 assessed with RT-qPCR and western blotting. (E) mRNA stability measurement by determining the TLR4 decay rate. Data were expressed as mean ± standard deviation. Normally distributed data were compared between two groups with the unpaired t-test and among multiple groups with one-way analysis of variance. Tukey’s test was used for post hoc analysis. *, P<0.05 compared with the control group; #, P<0.05 compared with the oe-NC group; &, P<0.05 compared with the si-NC group. Cell experiments were done three times. NC, negative control; FTO, fat mass and obesity-associated protein; TLR4, toll-like receptor 4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Dox, doxorubicin; RIP, RNA immunoprecipitation; m6A, N6-methyladenosine; RT-qPCR, reverse transcription quantitative polymerase chain reaction; Me-RIP, methylated-RIP.

Fig 5: m1A modification of SP100A increases its RNA stability and translational efficacy. (A) RIP-qPCR assay of SP100A expression in ocular melanoma cells (92.1, OMM2.3 and CRMM1) by YTHDF1, YTHDF2 and YTHDF3. The data are presented as the mean ± SD of experimental triplicates. Significance was determined by unpaired two-tailed Student's t test. **P < 0.01, ***P < 0.001. (B) Correlation analysis of SP100A expression and YTHDF1 expression in the TCGA-UM cohort (n = 80). Significance was determined by Pearson correlation analysis (R = 0.61, P < 0.0001). (C) qPCR data showing YTHDF1 RNA expression and SP100A RNA in ocular melanoma cells (92.1, OMM2.3 and CRMM1) upon YTHDF1 knockdown. The data are presented as the mean ± SD of experimental triplicates. Significance was determined by unpaired two-tailed Student's t test. ***P < 0.001, ****P< 0.0001. (D) Western blot showing YTHDF1, SP100A, ALKBH3 expression relative to ACTB expression in ocular melanoma cells (92.1, OMM2.3 and CRMM1) upon ALKBH3 and YTHDF1 knockdown. (E) The luciferase reporter gene assay demonstrated the relative luciferase activity of the wild-type and six mutant SP100A 5′UTR reporter vectors. The data are presented as the means ± SD of experimental triplicates. Significance was determined by unpaired two-tailed Student's t test. ****P < 0.0001. (F) Half-life of SP100A in wild-type and ALKBH3-deficient 92.1 cells treated with actinomycin (5 g/ml) for 0–6 h. (G) Polysome profiling assays of 92.1 cells with or without ALKBH3 knockdown. RNAs in different ribosome fractions were extracted and subjected to qPCR analysis. Data are shown as the mean ± SD. Significance was determined by unpaired two-tailed Student's t test. ***P < 0.001, ****P < 0.0001.