Fig 1: Expression of CCL5 and its receptor in different breast cancer cell lines and the secretion of CCL5. (A) The mRNA expression levels of CCL5, CCR1, CCR3 and CCR5 in different breast cancer cells; MCF-7 cells served as a control, while GAPDH expression was an internal reference. **P<0.01 vs. control group. (B) The protein expression of CCR5 was detected by western blotting, **P<0.01 vs. control group. (C) CCL5 secretion in different breast cancer cell lines using ELISA. **P<0.01 vs. control group. CCL5, C-C motif chemokine ligand 5; CCR1, C-C chemokine receptor type 1; CCR3, C-C chemokine receptor type 3; CCR5, C-C chemokine receptor type 5.
Fig 2: CS increases expression of HIV receptors CD4 and CCR5 in NHBE ALI cultures. NHBE ALI cultures were exposed to CS and total protein was extracted. Cells were lysed with RIPA buffer containing protease inhibitor cocktail and protein expression was quantified by Western blot analysis and normalized using β-actin. CS significantly enhances expression of CD4 (panel a) and CCR5 protein expression (panel b) when compared to air-exposed controls. CS does not increase CXCR4 protein expression (panel c). Western blot images (a–c) are representative of NHBE ALI cultures from three independent lungs. Relative density of the detected protein band was measured by using the ImageJ software and the values obtained were averaged. n = 3 lungs (unless stated otherwise). *Significant (p < 0.05).
Fig 3: Effect of CS exposure on CCR5 Cell surface receptor quantified by Single Cell Imaging flow cytometry. NHBE ALI cultures were exposed to CS. At the end of smoking, cells were immuno-stained with surface markers anti-human CCR5 labeled with APC. Percent Gated CCR5+ or APC+ cells for air NHBE (8.58 ± 2.85%) and smoke NHBE (20.25 ± 4.5%, P = 0.03) are represented (panel a). MFI of APC from in total population of cell images acquired (10,000 per sample) for air NHBE cells (818.74 ± 274.02) and smoked NHBE cells (1494.16 ± 447.95) are represented (panel b). Representative overlay histogram of intensity of APC for isotype control (yellow), air (blue) and smoke (green) (panel c). A representative single cell image for each sample is shown (panel d). Experiments were carried out from at least 3 different lungs. *Significant (p < 0.05).
Fig 4: CS increases CCR5 expression by TGF-ß mediated suppression of miR-141-5p. NHBE ALI cultures were exposed to CS. Total RNA was extracted and analyzed to quantify CCR5 mRNA expression by qRT-PCR. CS enhances CCR5 mRNA levels (panel a). Lung and age-matched NHBE ALI cultures were treated with recombinant TGF-ß1(10 ng/ml apically and basolaterally). After 20 hours, cells were lysed and total RNA was extracted and analyzed for CCR5 mRNA expression by qRT-PCR. TGF-ß1 increases CCR5 expression comparable to that observed in CS exposed cells (panel b). Total RNA was analyzed for expression of miR-141-5p from CS exposed and TGF-ß treated cells by qRT-PCR. We found the miR-141-5p expression is suppressed by CS as well as TGF-ß1 treatment (panels c,d). n = 3 different lungs. *Significant (p < 0.05).
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