Table 1.
Primer sequences used in quantitative real time PCR assays.
Fig 1.
Effect of heat shock on HSPs and m6A mRNA methylation related genes.
Expression of HSPA1B (HSP70) (A), HSPB1 (HSP27) (B), METTL3 (C), METTL14 (D), FTO (E), and YTHDF2 (F) mRNA at 6 h, 12 h, 24 h after heat shock in HepG2 cells. Data are shown as mean ± SEM (n = 3). *p value ≤ 0.05, **p value ≤ 0.01.
Fig 2.
Localization of YTHDF2 under heat shock.
The majority of YTHDF2 resided in the cytosol in normal conditions, whereas nearly all YTHDF2 translocated into the nucleus from the cytosol under heat shock stress. Scale bar = 88 μm.
Fig 3.
Effect of METTL3 knockdown on HSPs and cell viability in HepG2 cells.
Expression of METTL3 mRNA and protein in HepG2 cells after METTL3 knockdown (A and B). (n = 3). Expression of HSPA1B (HSP70), HSPA9 (HSP70), HSP90AA1 (HSP90), HSPD1 (HSP60), HSF1, and HSPB1 (HSP27) mRNA upon METTL3 knockdown in HepG2 cells (C) (n = 3). The relative cell viability determined by MTT at 24, 48, and 72 h post-transfection of METTL3 siRNA with or without heat shock pretreatment (D) (n = 6). Data are shown as mean ± SEM. *p value ≤ 0.05, **p value ≤ 0.01.
Fig 4.
Effect of METTL3 knockdown on the lifetime of HSPA1B in HepG2 cells.
Lifetime of HSPA1B (HSP70) mRNA in the samples following knockdown of METTL3 in HepG2 cells (A). The relative mRNA levels of HSPA1B (HSP70) in the samples following knockdown of METTL3 in HepG2 cells at 0 h, 3 h, and 6 h (B).
Fig 5.
Effect of YTHDF2 on HSPs mRNA expression and cell viability in HepG2 cells.
YTHDF2 knockdown decreased YTHDF2 mRNA in HepG2 cells (A). Expression of HSPA1B (HSP70), HSPA9 (HSP70), HSPB1 (HSP27), HSP90AA1 (HSP90), HSPD1 (HSP60) mRNA from the sample of YTHDF2 knockdown in HepG2 cells (B and C). The relative cell viability determined by MTT at 24, 48, and 72 h after knockdown of YTHDF2 with or without heat shock pretreatment (D) (n = 6). Data are shown as mean ± SEM. *p value ≤ 0.05, **p value ≤ 0.01.
Fig 6.
M6A methylated peaks of HSPs mRNA.
Integrative genomics viewer (IGV) plots showing m6A methylated peaks for HSPA1B (HSP70) (A), HSPB1 (HSP27) (B), HSPA9 (HSP70) (C), HSP90AA1 (HSP90) (D), HSPD1 (HSP60) (E), HSF1 (F) mRNA in HepG2 cells. Blue boxes represent exons and blue lines represent introns. n = 2.