Fig. 6: The effect of HIF1α on CCL5-induced HCC metastasis.

A The CAF-CM induced migration and invasion of Huh7 and Hep3B cells were abrogated after knockdown of HIF1α. Scale bar: 100 μm. B NOD/SCID mice were injected subcutaneously on the flanks with Huh7 cells or HIF1α knockdown (shHIF1α) Huh7 cells co-injected with CAFs (n = 5). Bouin’s liquid staining showed the pulmonary metastatic nodules (red arrows) from the subcutaneous xenograft tumors of mice (upper); H&E staining indicated tumor distribution (red arrows) in the lung (lower). Scale bar: 100 μm. C The CAF-CM induced the expression of EMT markers in Huh7 and Hep3B cells was suppressed by knockdown of HIF1α. D The expression of hydroxylated-HIF1α (HIF1α-OH 402 and HIF1α-OH 564) and prolyl hydroxylase (PHDs) in Huh7 and Hep3B cells treated with CAF-CM or CAF-CM by pre-incubation with a CCL5 neutralizing antibody was performed by western blotting. E Co-immunoprecipitation of ubiquitin and pVHL with HIF1α in Huh7 and Hep3B cells treated with CAF-CM or CAF-CM by preincubation with a CCL5 neutralizing antibody. F HIF1α Expression in cancer tissues and matching tumor-adjacent tissues of 20 liver cancer patients was detected by western blotting. G Survival analysis showed overall survival of patients with high and low levels of HIF1α (P = 0.0029, log-rank test, https://www.proteinatlas.org/ENSG00000100644-HIF1A/). N = 5 per group. Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the tumor control group. ##p < 0.01, compared with the Huh7+CAF group, ###p < 0.001, compared with the CAF-CM group.