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Fig. 6 | Molecular Cancer

Fig. 6

From: YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma

Fig. 6

HIF-2α blockade contributed to YTHDF2-dependet HCC inhibition. (a) Immunofluorescence staining of YTHDF2 and pimonidazole (PIMO) in SMMC7721-derived mouse tumors. Hypoxic tumor areas were marked by PIMO staining. n = 3 biological replicates. Scale bar, 20 μm.(b) Immunoblot of YTHDF2 in SMMC7721 cells transduced with a control siRNA (siCtrl) or siRNAs targeting HIF-1/2α and grown for 24 h under Nx or Hx. n = 2 independent experiments. (c) RT-qPCR analysis of YTHDF2 in SMMC7721 cells expressing indicated siRNAs after 24 h of Nx or Hx exposure. (d) Enrichment of hypoxia-responsive elements (HREs) -containing fragments of human Ythdf2 promoter in DNA-HIF-2α complex, as determined by ChIP-qPCR. n = 4 biological replicates. (e) Ythdf2 promoter activity in SMMC7721 cells expressing indicated siRNAs, as quantified using luciferase assay. Renilla luciferase activity was normalized to firefly activity and presented as relative luciferase activity. n = 3 biological replicates. (f) Proliferative activity of indicated SMMC7721 cells treated with vehicle or PT2385 (10 μM), as assessed by WST1 assay. n = 3 biological replicates. (g) Numbers of endothelial tubes formed by HUVEC cocultured with indicated conditions. n = 3 biological replicates. (h, j) Immunoblot of YTHDF2 (h) and p-STAT3 (j) in SMMC7721 cells treated with vehicle or PT2385 (10 μM). n = 2 independent experiments. (i) RT-qPCR analysis of IL11 (left) and SERPINE2 (right) mRNA levels. n = 3 biological replicates. (k, l) NPG mice bearing SMMC7721-shCtrl (K) or SMMC7721-shYTHDF2 (L) cells were orally treated with Vehicle or PT2385 (20 mg/kg/d) after reaching an average tumor volume of 200 mm3. Tumor volumes were continuously measured at indicated dates of treatment. n = 6 mice. Error bars indicate means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P-values were determined by two tailed t-test

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