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: YKL-40 and OLIG2 positivity delineates mesenchymal and proneural glioblastoma subtypes. (A) Strategy for subtyping of glioblastoma tumors. (B) Immunohistochemistry shows bright YKL-40-positivity regions (i) and regions-of-heterogeneity (ROH) showing coexpression of both YKL-40 and OLIG2 in tumor cells (ii). (C) Semiquantitative analysis of YKL-40 immunostained areas for glioblastoma subclass segregation. Percent-positive areas are calculated from data shown in B and are plotted as mean ± SEM from glioblastoma samples (n = 35), **p < 0.001. (D) YKL-40 and OLIG2 immunostaining in glioblastoma tumors (i), ROH as areas with cancer cells coexpressing both YKL-40 and OLIG2 proteins (marked areas) (ii). (E) Semiquantitative analysis of OLIG2 immunostained areas for glioblastoma segregation. Percent-positive areas are calculated from (D). Data are plotted as mean ± SEM from glioblastoma samples (n = 35), ****p < 0.0001. Magnification ×63. Sections were mounted in antifade mounting medium and acquired on a LSM 510, confocal microscope (Carl Zeiss AG).

Fig 4: Minimal multigene signature (MMS) glioblastoma effectively segregates preclassified tumors. (A) Immunostaining of MMS glioblastoma genes in representative glioblastoma tumors. (B) Derivation of immunohistochemistry score for each of the MMS glioblastoma genes in glioblastoma tumors. (C) Semiquantitative analysis of individual cancer cells differentially expressing each of the specific MMS glioblastoma genes. (D) Coexpression of YKL-40 and vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B) in representative glioblastoma tumors. (E) Single-cell profiling for coexpression of YKL-40 and each of the MMS glioblastoma genes. 1,000 tumor cells were plotted for both proneural and mesenchymal glioblastoma tumors (n = 10 each subtype). (F) Pearson correlation analysis of individual MMS glioblastoma proteins and YKL-40 in tumor regions. Individual MMS glioblastoma genes are positively correlated with YKL-40 expression (VEGF-A, r = 0.9042; VEGF-B, r = 0.9751; angiopoietin 1 (ANG1), r = 0.6596; and angiopoietin 2 (ANG2), r = 0.7850).

Fig 5: Effects of relaxin (R) in the NF-κB knockout conditions. Knockout of NF-κB (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-β (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 α-SMA (α-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.