Fig 2: siRNA c-Myc knockout effect on Relaxin (R) in rhinovirus-infected cells. c-Myc transcription factor knockout inverted the relaxin (R) effect of a decrease in MMP-9 (Matrix metalloproteinase 9, (A)) expression. TGF-ß (B), and collagen I (C) mRNA expression was significantly decreased by relaxin. Under the conditions used, no effect of relaxin or rhinoviruses were observed in aDAM33 (D), YKL-40(Chitinase-3-like protein 1) (E) and LTC4S (G) expression. Relaxin changed the expression of RXFP1 in epithelial cells (F), and in fibroblasts in a-SMA (H). No difference was noticed between the specimens when relaxin was added to HRV (Human Rhinovirus)-infected cells (H16R, H2R) or prior to the HRV infection. H16R, H2R—rhinovirus 16 and 2 added first, followed by relaxin—after 24 h, as well as RH16, RH2—relaxin added first, followed by rhinovirus 16 and 2—added after 24 h. The data presented are normalized to the samples treated with control siRNA. * p < 0.05 in comparison to the control sample, and + p < 0.05 in comparison to the rhinovirus sample (HRV-2 or HRV-16, respectively); C is the control sample with medium only. N = 6, error bars—SEM.

Fig 3: Real-time PCR results presenting the effect of relaxin on the genes contributing airway remodeling after STAT3 siRNA silencing. The knockdown of STAT3 reversed relaxin’s (R) ability to influence MMP-9 (Matrix metalloproteinase 9, (A)), TGF-ß (Transforming growth factor beta, (B)), COL I (collagen I, (C)), and a-SMA (a-smooth muscle actin, (H)) mRNA expression. The knockout did not change levels of ADAM33 (ADAM Metallopeptidase Domain 33, (D)), YKL-40 (E) and LTC4S (leukotriene C4 synthase, (G)) mRNA expression in comparison to cells expressing STAT3. Relaxin increased mRNA expression of RXFP1 (F). Moreover, no difference were observed between the specimens when relaxin was added to cells infected with rhinovirus-16 (H16) and rhinovirus-2 (H2) or prior to the HRV infection. H16R, H2R—rhinovirus 16 and 2 added first, followed by relaxin—after 24 h, as well as RH16, RH2—relaxin added first, followed by rhinovirus 16 and 2—added after 24 h. The data presented are normalized to the samples treated with control siRNA. * p < 0.05 in comparison to the control sample, and + p < 0.05 in comparison to the rhinovirus sample (HRV-2 or HRV-16, respectively); C is the control sample with medium only. N = 6, error bars—SEM.

Fig 4: Effects of relaxin (R) in the NF-?B knockout conditions. Knockout of NF-?B (nuclear factor kappa-light-chain-enhancer of activated B cells) resulted in a lack of changes in MMP-9 (Matrix metalloproteinase 9) mRNA expression, either after relaxin or after HRV-2 (Human Rhinovirus) and HRV-16 treatment (A). Additionally, rhinovirus infections did not result in changes in the expression of MMP-9 (Matrix metalloproteinase 9, (A)), and TGF-ß (Transforming growth factor beta, (B)) mRNA expression. Similarly, no significant effects of relaxin were observed in the mRNA expression of collagen I (C). No effects of relaxin have been observed in ADAM33 (D), and YKL-40 (Chitinase-3-like protein 1, (E)) mRNA expression, while relaxin increased mRNA expression of RXFP1 (F). In case of LTC4S (G) and a-SMA (a-smooth muscle actin, (H)), relaxin did not cause significant change of mRNA expression. No difference has been observed between the specimens when relaxin was added to HRV infected cells or prior to the HRV infection. H16R, H2R—rhinovirus 16 and 2 added first, followed by relaxin—after 24 h, as well as RH16, RH2—relaxin added first, followed by rhinovirus 16 and 2—added after 24 h. The data presented are normalized to the samples treated with control siRNA. * p < 0.05 in comparison to the control sample, and + p < 0.05 in comparison to the rhinovirus sample (HRV-2 or HRV-16, respectively); C is the control sample with medium only. n = 6, error bars—SEM.