Figure 3. Mechanosensitive PANX1 channels release ATP to increase cancer cell survival during intravascular membrane stretch.

a, Representative images of mouse lungs stained for cancer cells and blood vessels (green and magenta, respectively, top panel), blood vessels (black, middle panel) or cleaved caspase-3 (white, bottom panel) 3 hrs after tail-vein injection of 1 × 10⁵ CN-LM1A cells. Arrows indicate endothelium. Scale bar, 10 µm. b, Quantification of PANX1-mediated CBX-sensitive ATP release from CN-LM1A and MDA-LM2 cells during 5 min exposure to isotonic (100% PBS) or hypotonic (70% PBS) solution; n = 4 (CN-LM1A isotonic), n = 3 (CN-LM1A hypotonic), n = 4 (MDA-LM2 isotonic), n = 4 (MDA-LM2). c, Quantification of viable, trypan blue-negative, CN34 and CN-LM1A cells after 1 hr extreme hypotonic (12.5% PBS) stretch; n = 4. d, Quantification of viable, trypan blue-negative CN34, CN-LM1A, MDA-MB-231 and MDA-LM2 cells after 1 hr extreme hypotonic (12.5% PBS) stretch; n = 4. e, Quantification of viable, trypan blue-negative, CN-LM1A cells after 1 hr incubation in extremely hypotonic (12.5% PBS) solution in the presence of scrambled peptide (100 µM), ¹⁰Panx1 peptide (100 µM) or ¹⁰Panx1 peptide (100 µM) and 100 µM ATP; n = 4. f, Quantification of viable, trypan blue-negative, MDA-LM2 cells after 1 hr incubation in extremely hypotonic (12.5% PBS) solution in the presence of scrambled peptide (100 µM), ¹⁰Panx1 peptide (100 µM) or ¹⁰Panx1 peptide (100 µM) and 100 µM ATP; n = 4. g, Quantification of viable, trypan blue-negative MDA-LM2 cells with 30 min Boyden chamber centrifugation (3,800 rpm) after cells were pre-treated for 10 min with 500 µM CBX or PBS; n = 4. Error bars, s.e.m., *, P < 0.05; **, P < 0.01; ***, P < 0.001 by a one-tailed Student’s t-test. n represents biological replicates. Experiments b–g are representative and were replicated at least two times with two independent cell lines.