Figure 6.
Loss of METTL1 significantly suppresses intrahepatic metastasis of HCC cells after sublethal heat stress in vivo
(A) Flow chart of the construction of intrasplenic injection model. (B and C) Representative images (B) of liver metastasis in control and METTL1 knockdown HCC cells with or without heat treatment (N = 7). Blue arrow: liver metastasis. Quantitative comparison of mice with liver metastasis after intrasplenic injection of heat-treated or control HCC cells (C). (D) Quantitative comparison of tumor burden by calculating liver weight to body weight ratio of each mouse. (E and F) Representative images (E) and quantification data (F) of liver metastasis in each group with or without heat treatment by H&E staining. (G–I) Analysis of expression levels of METTL1/WDR4 (G) and SLUG/SNAIL (I) by WB, and m7G tRNA modification level (H) by northwestern blot in HCC metastatic tissue with or without heat treatment. (J–M) Representative images (J) and quantification data of METTL1 (K), WDR4 (L), and Ki-67 (M), all of which were detected higher in group with sublethal heat stress by IHC staining. U6 snRNA was used as a loading control for northwestern blot. β-Actin was used as a loading control for WB. Scale bars: 200 μm and 50 μm. Data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (Student’s t test, one-way ANOVA). shNC, negative control; sh1, shMETTL1-1; sh2, shMETTL1-2; HE, hematoxylin and eosin staining; IHC, immunohistochemistry staining; WB, western blot.