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. 2021 Dec 12;11(12):e661. doi: 10.1002/ctm2.661

FIGURE 6.

FIGURE 6

Liver‐specific knockout of Mettl1 inhibits HCC tumourigenesis in vivo. (A) The schematic diagram of the construction of Mettl1 conditional liver‐specific knockout mice. (B) Representative image of livers harvested from Mettl1‐cKO and control group. (C) Comparison of ratio of liver weight to body weight between Mettl1‐cKO and control group. Data presented as mean ± SD (n = 5). (D) Representative images of H&E staining of control and Mettl1‐cKO mouse livers. Scale bar: 100 μm. (E) IHC staining of Ki‐67 in control and Mettl1‐cKO mouse livers. Arrows point to Ki67‐positive cells. Scale bar:100 μm. Data presented as mean ± SD (n = 5). (F) General view of hydrodynamics transfection experiment in Mettl1 conditional knockout mice and control. (G) Representative image of livers harvested from Mettl1‐cKO and control group. (H) Comparison of tumour burden between Mettl1‐cKO and control group by ratio of liver weight to body weight (LW/BW). Data presented as mean ± SD (n = 9). (I) Representative images of H&E staining of control and Mettl1‐cKO mouse livers. Arrows point to tumour lesions. Scale bar: 250 μm. The number of tumour foci in these mouse livers was evaluated. Data presented as mean ± SD (n = 9). (J) Left panel, representative images of IHC staining of Ki‐67. Arrows point to Ki67‐positive cells. Scale bar:100 μm. Right panel, the statistical analyses. Data presented as mean ± SD (n = 9). (K) Western blot of Mettl1, EGFR, Cyclin A2 and AFP in tumour tissues from control group and Mettl1‐cKO group. β‐Actin was used as a loading control. Downregulation of m7G tRNA modification and LysCTT in Mettl1‐cKO group was confirmed by northwestern blot and northern blot. U6 was used as a loading control. (L) qRT‐PCR analysis of Cyclin A2 and EGFR mRNA levels in tumour tissues from control group and Mettl1‐cKO group. Data presented as mean ± SD (n = 3). *p < .05, **p < .01, ***p < .001 by Student's t test, one‐way ANOVA or the Mann–Whitney U test unless specified. All the in vitro assays were biologically repeated for three times. Abbreviations: cKO, conditional knockout; Ctrl, control