Fig 1: CAFs secrete elevated CCL5 to promote cell viability in NSCLC cancer cells via paracrine activation. (A) mRNA expression levels of CCL5 in CAFs, NFs and two NSCLC cell lines by RT-qPCR using GAPDH gene as the internal control. (B) CCL5 in conditioned medium secreted by NFs and CAFs were quantified by ELISA. *P<0.05, ***P<0.001 and ****P<0.0001 vs. CAFs. (C) A549 and H1299 cancer cells were incubated with CAF-CM or NF-CM in combination with anti-CCL5 antibody (0.1 µg/ml) for 6 h followed by treatment with DDP for 48 h. Cell viability was determined by the MTT assay. (D) mRNA expression levels of CCL5 and CCR5 in NSCLC cell lines cultured with CAF-CM were assessed by RT-qPCR using GAPDH gene as the normalization control. (E) A549 and H1299 cancer cells were incubated with CAF-CM or NF-CM in combination with CCR5 inhibitor (Met-RANTES; 0.1 µg/ml) for 6 h followed by treatment with DDP for 48 h. Cell viability was determined by the MTT assay. *P<0.05, **P<0.01 and ***P<0.001 vs. control; #P<0.05 and ##P<0.01 vs. DDP; &&P<0.01 vs. CAF-CM and DDP. Results were expressed as the mean ± SD, and the means were calculated from =3 independent experiments. RT-qPCR, reverse transcription-quantitative PCR; CAF, cancer-associated fibroblast; NF, normal fibroblast; CM, conditioned medium; DDP, cisplatin; CCL5, C-C motif chemokine ligand 5; CCR5, C-C motif chemokine receptor 5; NSCLC, non-small cell lung cancer.
Fig 2: CAF-CM increases HOTAIR expression in tumor cells in vitro. (A) HOTAIR expression in human NSCLC A549 and H1299 cell lines treated with CAF-CM or NF-CM was determined by qPCR. HOTAIR expression in human NSCLC A549 and H1299 cell lines treated with CAF-CM and (B) CCL5 neutralizing antibody or (C) CCR5 inhibitor (Met-RANTES) was determined by qPCR. ***P<0.001 and ****P<0.0001 vs. control; ##P<0.01 and ###P<0.001 vs. CAF-CM. Results were expressed as the mean ± SD, and the means were calculated from =3 independent experiments. qPCR, quantitative PCR; CAF, cancer-associated fibroblast; NF, normal fibroblast; CM, conditioned medium; CCL5, C-C motif chemokine ligand 5; CCR5, C-C motif chemokine receptor 5; NSCLC, non-small cell lung cancer; HOTAIR, HOX transcript antisense RNA.
Supplier Page from Abcam for Human RANTES ELISA Kit (CCL5)