Figure 3(A) N6-methyladenosine (m6A) modification levels of mRNAs in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction, detected by m6A RNA Methylation Quantification Kit (Colorimetric) (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test (A *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(B) qRT-PCR analysis of the relative mRNA levels of m6A writers (METTL3, METTL4, METTL14 and WTAP) and erasers (FTO and ALKBH5) in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction (n = 6-9). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test B, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(C) Western blotting analysis of m6A writers and erasers in normal and EP3-deficient brown pre-adipocytes with or without brown adipogenesis induction.(D) Quantification of protein levels in C (n = 4-6). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates , D, Statistical significance was evaluated by unpaired two-tailed student's t test D, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(E) Western blotting analysis of WTAP in iBAT from EP3F/F and EP3F/FMyf5Cre mice fed HFD for 1 week.(F) Western blotting analysis of WTAP in BAs differentiated from EP3γ-expressed brown pre-adipocytes.(G) Relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed brown pre-adipocytes detected by RT-qPCR (n = 6-7). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates G, Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons (G, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(H) qRT-PCR analysis of the relative mRNA levels of EP3γ gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). (I) qRT-PCR analysis of the relative mRNA levels of WTAP gene in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by unpaired two-tailed student's t test H), two-way ANOVA followed by Tukey's test for post-hoc comparisons I, *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).(J) Representative picture of oil red O-stained BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes with WTAP siRNA treatment. Scale bar: 50 μm.(K-Q) qRT-PCR analysis of the relative mRNA levels of brown marker genes in BAs differentiated from EP3γ-expressed EP3-/- brown pre-adipocytes after transfection with WTAP siRNA (n = 6-8). Data information: Data represent the mean ± standard error of mean (SEM). Data are representative of two independent experiments with biological replicates Statistical significance was evaluated by two-way ANOVA followed by Tukey's test for post-hoc comparisons K-Q). *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant (P > 0.05).