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.
Fig 3: Co-stimulation of Nrf2 and DKK1 signaling boosts the anti-stress capacity of human MSCs. (A) Left: Changes in Nrf2 activity in MSCs with or without TNF-α/H2O2 co-treatment. Right: Representative immunoblot results for DKK1 and secreted DKK1 (sDKK1) in MSCs with a similar test design as in (A) (n = 6). (B) ELISA results of released IL-1β, IL-6, MCP-1, RANTES, and IL-10 protein from MSCs treated with TNF-α/H2O2 (n = 6). (C) Changes in cell viability and apoptotic ratios of MSCs following challenge with TNF-α/H2O2 in the presence or absence of Nrf2/DKK1 manipulations (n = 6). (D) Representative immunoblot results for PCNA and Bcl-2 from MSCs following the aforementioned treatments. (E) Changes in cellular caspase-3/8 activity of MSCs with the aforementioned treatments (n = 6). (F) Changes in mitochondrial superoxide levels in MSCs measured by MitoSOX in flow cytometry following the aforementioned treatments. (G) Fluorescence micrographs for the detection of cellular ROS by CellROX Green and corresponding quantified fluorescence intensities in MSCs treated with TNF-α/H2O2 with the aforementioned treatments (n = 6). Scale bar = 50 μm. hADMSCs = human adipose-derived mesenchymal stromal cells. Values are expressed as mean ± SD. *, **, and *** indicate P < 0.05, 0.01, and 0.001 against an untreated MSC group (or between indicated groups), respectively; #, ##, and ### indicate P < 0.05, 0.01, and 0.001 against TNF-α/H2O2-treated control group, respectively.
Supplier Page from Abcam for Human RANTES ELISA Kit (CCL5)