Table 1.
Antibodies used in the study.
Fig 1.
Proliferation of RAW 264.7 cells by direct cell counting with hematoxylin staining.
10 μg/mL PTX-treated RAW 264.7 cells showed the increase of proliferation by 3.5% at 12 h, 6.7% at 24 h, and 11.2% at 48 h compared to the non-treated controls. A-D: Histological observation at x400 magnification, E: Statistical analysis plotted into a rod graph, cell number versus culture time (0, 12, 24, and 48 h).
Fig 2.
Immunocytochemical staining of Ki-67 (A), KMD4D (B), PCAF (C), TNFα (D), IL-6 (E), TLR3 (F), and TLR4 (G) in RAW 264.7 cells after 10 μg/mL PTX treatment for 0, 12, 24, and 48 hours. Noted the cytoplasmic (arrow heads) and nuclear (arrows) localization of different immunoreactions in monocytic round cells. No counter stain.
Fig 3.
Immunocytochemical staining of GSTO1/2 (A), LC3β (B), GADD153 (C), PARP-1(D), and caspase-3 (E) in RAW 264.7 cells after 10 μg/mL PTX treatment for 0, 12, 24, and 48 hours. Noted the cytoplasmic (arrow heads) and nuclear (arrows) localization of different immunoreactions in monocytic round cells. No counter stain.
Fig 4.
Immunocytochemical staining of NSEγ (A), MYH2 (B), TGF-β1 (C), RUNX2 (D), OPG (E), and BMPR2 (F) in RAW 264.7 cells after 10 μg/mL PTX treatment for 0, 12, 24, and 48 hours. Noted the cytoplasmic (arrow heads) and nuclear (arrows) localization of different immunoreactions in monocytic round cells. No counter stain.
Fig 5.
Western blot analysis for 10 μg/mL PTX-induced protein expression in RAW 264.7 cells regarding the proliferation (Ki-67), p53/Rb/E2F signaling (p53, Rb1, and E2F1), Wnt/β-catenin signaling (Wnt1, β-catenin, and TCF1), guided cell migration (E-cadherin and VE-cadherin), cMyc/MAX/MAD network (cMyc, MAX, and MAD1), epigenetic modification (KDM4D and HDAC10), and RAS signaling (KRAS, HRAS, NRAS, ERK1, and p-ERK1).
The level of β-actin expression was used as an internal control.
Fig 6.
Western blot analysis for 10 μg/mL PTX-induced protein expression in RAW 264.7 cells regarding the inflammation (TNFα, TLR2, and TLR4), ER stresses (eIF2AK3, p-eIF2AK3, ATF4, LC3β, and caspase 3), osteoblastic differentiation (TGF-β1, BMP2, and RUNX2), neurogenic differentiation (NSEγ and NF1), and muscular differentiation (MYH2 and desmin).
The level of β-actin expression was used as an internal control.
Fig 7.
Western blot comparison between 10 μg/mL and 300 μg/mL PTX-induced protein expressions in RAW 264.7 cells regarding the proliferation (Ki-67, cMyx, MAX, E2F1, Wnt1, and TCF1), epigenetic modification (HDAC10), RAS signaling (KRAS, ERK1, and pERK1), apoptosis (p53 and c-caspase 3), ER stresses (eIF2AK3, p-eIF2AK3, and ATF4), inflammation (TNFα and TLR2), and osteogenesis (BMP2, and RUNX2).
The level of β-actin expression was used as an internal control.
Fig 8.
Expression of proliferation-related proteins (A and B), cMyc/MAX/MAD network proteins (C and D), and double IP-HPLC for cMyc/MAX/MAD network protein complexes (E and F) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 9.
Expression of p53/Rb/E2F signaling proteins (A and B), double IP-HPLC for p53/Rb/E2F signaling protein complexes (C and D), Wnt/β-catenin signaling proteins (E and F), and double IP-HPLC for Wnt/β-catenin signaling protein complexes (G and H) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 10.
Expression of epigenetic modification proteins (A and B), protein translation proteins (C and D), and growth factors (E and F) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 11.
Expression of RAS signaling proteins (A and B), NFkB signaling proteins (C and D), and upregulated inflammatory proteins (E and F) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 12.
Expression of downregulated inflammatory proteins (A and B), p53-mediated apoptosis proteins (C and D), and FAS-mediated apoptosis proteins (E and F) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 13.
Expression of protection-related proteins (A and B), survival and aging-related proteins (C and D), endoplasmic reticulum stress proteins (E and F), and SHH/PTCH/GLI and Notch/Jagged signaling proteins (G and H) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, E, and G show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, F, and H) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 14.
Expression of cytodifferentiation proteins (A and B), neuromuscular differentiation proteins (C and D), and fibrosis proteins (E and F) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 15.
Expression of oncogenesis proteins (A and B), angiogenesis proteins (C and D), and osteogenesis proteins (E and F) in 10 μg/mL PTX-treated RAW 264.7 cells as determined by IP-HPLC. Line graphs, A, C, and E show protein expression on the same scale (%) versus culture time (12, 24, or 48 h), whereas the star plots (B, D, and F) show the differential expression levels of the proteins at 12, 24, or 48 h after PTX treatment on the appropriate scales (%). The thick black line, untreated controls (100%); the blue, yellow, and red dots show differential protein levels after PTX administration for 12, 24, or 48 h, respectively.
Fig 16.
Star plot of global protein expression in RAW 264.7 cells treated with 10 μg/mL PTX.
The representative proteins (n = 150) were selected and their maximum or minimum expression levels (%) were plotted in a circular manner. 21 major signaling pathways showed different levels of protein expression. 10 μg/mL PTX activated the growth factors, epigenetic modification, protein translation, RAS and NFkB signaling, protection, neuromuscular ad osteoblastic differentiation, acute inflammation associated with innate immunity and cell-mediated immunity, but inactivated ER stress, fibrosis, and chronic inflammation. FAS-mediated apoptosis was enhanced contrary to p53-mediated apoptosis. Also noted that the upregulation of oncogenic proteins and the downregulation of tumor suppressor proteins. Red circle: maximum expression of upregulated proteins. Blue circle: minimum expression of downregulated proteins.
Fig 17.
IP-HPLC comparison between the protein expression of 10 μg/mL and 300 μg/mL PTX-treated RAW 264.7 cells.
Expression of proliferation-related proteins (A and B), RAS/NFkB signaling proteins (C and D), inflammatory proteins (E and F), and apoptosis proteins (G and H) as determined by IP-HPLC. Line graphs, A, C, E, and G show 10 μg/mL PTX-induced protein expressions, whereas B, D, F, H show 300 μg/mL PTX-induced protein expressions on the same scale (%) versus culture time (12, 24, or 48 h).
Fig 18.
IP-HPLC comparison between the protein expression of 10 μg/mL and 300 μg/mL PTX-treated RAW 264.7 cells.
Expression of ER stress proteins (A and B) fibrosis proteins (C and D), neuromuscular differentiation proteins (E and F) and osteogenesis proteins (G and H) as determined by IP-HPLC. Line graphs, A, C, E, and G show 10 μg/mL PTX-induced protein expressions, whereas B, D, F, H show 300 μg/mL PTX-induced protein expressions on the same scale (%) versus culture time (12, 24, or 48 h).
Fig 19.
A diagram of 10 μg/mL PTX-induced protein expression change in global protein signaling pathways of RAW 264.7 cells.
The main axis of cellular signaling, that is, proliferation, RAS signaling, NFkB signaling, protection, survival and aging, and inflammation were consistently activated by 10 μg/mL PTX, and subsequently followed by activation of epigenetic modification, protein translation, Wnt/β-catenin, cMyc/MAX/MAD network, neuromuscular and osteoblastic differentiation, acute inflammation, innate immunity, cell-mediated immunity, and FAS-mediated apoptosis, while inactivation of p53/Rb/E2F signaling, ER stress, angiogenesis, fibrosis, and chronic inflammation. Sky blue letter: downregulated expression (under 80%), Blue letter: downregulated expression (80–95%), Green letter: minimal change expression (95–105%), Red letter: upregulated expression (105–120%), Flower pink letter: upregulated expression (over 120%).