Fig 2: Immunocytochemistry results indicating nuclear localization of PRP-1 and nuclear colocalization with MUC5B, TLR1, TLR6 in human chondrosarcoma JJ012 cell line. (A) PRP-1 antibody localization in nucleus of human chondrosarcoma JJ012 cells. Dark blue color-DAPI was applied for nuclear staining. Zenon Alexa fluor rabbit 488IgG (green) was used for PRP-1 rabbit serum IgG antibody staining. (B) MUC5B receptor localization in the nucleus of human JJ012 chondrosarcoma cells. Alexa Fluor 594 wheat germ agglutinin (WGA) was used to label plasma membrane (red) at 1:200 dilution for 1 h. DAPI (dark blue) stained nucleus at 3 µM. Rabbit anti-MUC5B was adopted as primary, and green goat anti-rabbit IgG H&L (DyLight 488) was used as a secondary antibody. (C) Composite image of nuclear localization of MUC5B (left panel) and PRP-1 antibody (right panel) in human JJ012 chondrosarcoma cells. Dark blue color-DAPI was applied for nuclear staining. Zenon Alexa Fluor rabbit 488IgG (green) was used for PRP-1 rabbit serum IgG antibody detection. Rabbit anti-MUC5B was adopted as primary, and green goat anti-rabbit IgG H&L (DyLight 488) was used as a secondary antibody. (D) TLR1 receptor localization in the cytoplasm and the nucleus in human chondrosarcoma JJ012 cells. TLR1 rabbit antibody was used as a primary, whereas goat anti-rabbit H&L (DyLight 550) was applied as the secondary antibody (yellow color). (E) TLR6 receptor nuclear and cytoplasmic colocalization with PRP-1 is demonstrated, H&L DyLight 550 was used as a secondary antibody (yellow). PRP-1 was stained with Zenon Alexa Fluor 488 IgG (green). (F) Western blot of nucleolin protein expression in human chondrosarcoma cell line. PRP-1 did not have any effect on nucleolin expression, indicating the absence of PRP-1 location in nucleoli. The band corresponded to MW of 77 kDa.

Fig 3: PRP-1 effect on TLR receptors and adaptor proteins in human chon-drosarcoma JJ012 cell line. PRP-1 upregulated protein expression of TLR1 and TLR2 in dose-dependent manner in human JJ012 chondrosarcoma cell lysates. TLR3 expression was very weak and the data is not shown. TLR4, TLR5 proteins were expressed but PRP-1 did not have any effect. Tubulin was used as loading control. The bands were detected for TLR1, TLR2, TLR4 and TLR5 and tubulin at 120, 119, 130, 90 and 50 kDa, correspondingly. PRP-1 upregulated protein expression of TLR6 in dose-dependent manner in human JJ012 chondrosarcoma cell lysates. TLR7 was not expressed in this cell line at all. TLR8, 9 and 10 were expressed but no effect of PRP-1 was observed. Tubulin was used as housekeeping control. The bands were detected for TLR6, TLR8, TLR9, TLR10 and tubulin at 100, 86, 100, 120 and 50 kDa, correspondingly. PRP-1 upregulated TICAM2 (TRAM) adaptor protein in dose-response manner but did not have any effect on TICAM1 (TRIF) adaptor protein in human JJ012 chondrosarcoma cell line. Tubulin was used as loading control. The bands were detected for TICAM1, TICAM2 and tubulin.

Fig 4: Heat map of gene expression (qRT PCR) upon PRP-1 treatment. The clustrogram performs non-supervised hierarchical clustering of the entire dataset to display a heat map with dendrograms indicating co-regulated genes across groups or individual samples; it represents the average of Ct values displayed across the genes of each sample. S2 stands for samples treated with 1 µg/ml of PRP-1 and S1 stands for 10 µg/ml of PRP-1. TLR2 and TLR6 were highly expressed in S2, while TLR1 was highly expressed in S1 and moderately in S2 group. c-Myc and MUC5B were highly expressed in S2. GAPDH and ACTB were housekeeping genes.