Figure 2. Long-term effect of heat stress on m6A methylation of EZH2, BDNF, and HSP70 transcripts in the AH. A–C, m6A enrichment along the EZH2 (A), BDNF (B), and HSP70 (C) transcripts in the AH during heat challenge on day 10. Total RNA was isolated from the AH of 10-d-old chicks before (0 h) and 6 h into heat challenge and then subjected to m6A-RIP with anti-m6A antibody followed by real-time PCR. The levels of EZH2, BDNF, and HSP70 mRNA in the RIP samples were normalized against the HMBS ones (EZH2 m6A-IP, BDNF m6A-IP, and HSP70 m6A-IP, specifically). Each value is the mean ± SEM of 9–13 pools of the two chicks normalized to that of non-conditioned chicks (non-cond) at 0 h, which was set to 1. For EZH2 m6A-IP conditioning effect F(2,54) = 0.111, p = 0.89; challenge effect F(1,54) = 0.045, p = 0.83; interaction F(2,54) = 3.885, p = 0.026; two-way ANOVA. For BDNF m6A-IP conditioning effect F(2,54) = 0.297, p = 0.74; challenge effect F(1,54) = 0.102, p = 0.75; interaction F(2,54) = 6.196, p = 0.004; two-way ANOVA. Fisher’s LSD multiple comparisons tests: # significant difference between 36°C‐ and 40°C‐conditioned chicks (36°C-cond and 40°C-cond, respectively); * significant difference relative to respective control (0 h). D–F, mRNA levels of EZH2 (D), BDNF (E), and HSP70 (F) in the AH during heat challenge on day 10. Total RNA from the AH of the same samples described in A–C was subjected to real‐time PCR with EZH2 (D), BDNF (E), and HSP70 (F) primers, respectively. HMBS mRNA expression was used as the standard. Results are mean ± SEM of 20–25 chicks in each group. The PCR relative value of non-conditioned chicks at 0 h was set to1. For EZH2 mRNA conditioning effect F(2,129) = 2.97, p = 0.055; challenge effect F(1,129) = 10.47, p = 0.001; interaction F(2,129) = 2.98, p = 0.054; two-way ANOVA. For BDNF mRNA conditioning effect F(2,128) = 2.49, p = 0.086; challenge effect F(1,128) = 0.861, p = 0.35; interaction F(2,128) = 2.515, p = 0.085; two-way ANOVA. For HSP70 mRNA conditioning effect F(2,129) = 14.06, p < 0.0001; challenge effect F(1,129) = 158, p < 0.001; interaction F(2,129) = 3.54, p = 0.032; Fisher’s LSD multiple comparisons tests: * significant difference relative to respective control (0 h); # significant difference in HSP70 mRNA level between 36°C‐ and 40°C‐cond at 6 h into the challenge. G–I, m6A enrichment along the EZH2 (G), BDNF (H), and HSP70 (I) transcripts in the FB during heat challenge on day 10. J–L, m6A enrichment along the EZH2 (J), BDNF (K), and HSP70 (L) transcripts in the IMM during heat challenge on day 10. Total RNA isolated from the FB (G–I) and IMM (J–L) was subjected to m6A-RIP with anti-m6A antibody followed by real-time PCR as described in A–C. Each value is the mean ± SEM of six pools of the two chicks normalized to that of non-conditioned chicks (non-cond) at 0 h, which was set to 1.