Figure 5

Figure 5

(a) Schema of experimental protocol for analysis of gene expression in lung alveolar macrophages at day 1 after naphthalene-induced epithelial injury in Panx1+^/+ and Panx1^-/- mice. (b) qPCR analysis of Arg1, Uap1, Sgk1 and Areg in naïve and post-naphthalene injury alveolar macrophages from Panx1+^/+ and Panx1^-/- mice (n-3-4/group, one experiment, assessed by one-way ANOVA with Tukey's multiple comparisons test). (c) Analysis of amphiregulin protein in BALF supernatant in Panx1+^/+ and Panx1^-/- mice at day 1 after epithelial injury, analysed by Luminex (n=4-5/group, one experiment, assessed by t-test). (d) Schematic for neutralising amphiregulin in vivo by administration of anti-amphiregulin antibody (5μg in 200μL sterile PBS given intraperitoneally) on days 0,1,2,3,4,&4.5 after naphthalene injury, with epithelial proliferation assessed by EdU incorporation (n=7/group, two independent experiments, assessed by t-test). (e) Schema of experimental protocol for inducing apoptosis in Panx1+^/+ or Panx1^-/- BEAS-2B epithelial cells with (f) confirmation of apoptosis induction (AnnV/7AAD staining) and Panx1 channel opening in Panx1+^/+ apoptotic cells (AnnV/TO-PRO staining) and (g) apoptotic cell supernatant transferred onto mouse bone marrow derived macrophages (BMDMs) for 4h prior to qPCR for Areg (n=5, assessed by oneway ANOVA with Holm-Šídák's multiple comparisons test). (h) BMDMs were treated with increasing concentrations of the Panx1-released nucleotide ATP for 4h prior to qPCR for Areg (n=4, assessed by one-way ANOVA with Holm-Šídák's multiple comparisons test, each group compared to control/0μM ATP). (i) Degradation of extracellular ATP using recombinant CD39/ENTPD1 (rCD39; 44ng/ml) attenuates induction of Areg by BMDMs in response to apoptotic epithelial cell supernatants (qPCR after 4h; n-=4, assessed by one-way ANOVA with Holm-Šídák's multiple comparisons test). *p<0.05, **p<0.01. *** p<0.001, **** p<0.0001.