Fig 2: CS-6 suppressed the protein expression of the OC marker genes. (A) The results of the protein expression of OC-related genes including TRAF6, MMP-9, c-Fos, and NFATc1 in BMM cells stimulated with RANKL (50 ng/ml) with indicated concentrations of CS-6 (100 nM) for 5–7 days compared to the control group (30 ng/ml MCSF only). (B) The expression of related protein was quantified. (C) The results of the protein expression of OC-related genes including TRAF6, MMP-9, c-Fos, and NFATc1 in BMM cells stimulated with RANKL (50 ng/ml) for 3 days prior to indicated concentrations of CS-6 (100 nM) compared to the control group (30 ng/ml MCSF only). (D) The expression of related protein was quantified. Values were presented as the mean ± standard deviation (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Overexpression of FOXM1 stimulated UV irradiation-mediated dysfunction of keratinocytes. (a) Vectors and FOXM1 overexpression plasmids were transfected into HaCaT cells, and the transfection efficiency was inspected by RT-PCR after 24 hours. The transfected HaCaT cells were then subjected to UV irradiation and treated with 5 μM curcumin for 24 hours. (b) UV-irradiated HaCaT cell proliferation was assayed using CCK-8. (c)–(e) Levels of SOD, GSH-PX, and MDA in UV-irradiated HaCaT cells were compared using the Oxidative Stress Assay Kit. (f) The amount of ROS in UV-irradiated HaCaT cells was determined by cytofluorimetry. (g) ELISA was implemented to verify the profiles of inflammatory factors (IL-1β, IL-6, IL-18, TNFα) in UV-irradiated HaCaT cells. (h)–(k) Expression of Bax, Bcl-xL, Caspase3, Caspase8, Caspase9, Keap1, Nrf2, HO-1, COX2, iNOS, NF-κB, MMP1, MMP9, SPAG5, and FOXM1 in UV-irradiated HaCaT cells was examined with WB. N = 3. ∗p < 0.05 (vs. vector or UV group), ∗∗p < 0.01, ∗∗∗p < 0.001; #p < 0.05 (vs. FOXM1+UV group), ##p < 0.01, ###p < 0.001.

Fig 4: ß5 Integrin/MMP-9 axis regulates acute lung injury in vivo. (A) Immunoblot analysis of ß5 Integrin and MMP-9 from mice intratracheally treated with control or LPS for 18 h. Data and means ± SEM of 4 mice per group. (B) Linear regression of Itgb5 gene expression normalized to control mice vs BALF MMP-9 in ALI subjects (n=12). (C) EBA uptake, (D) protein concentrations, (E) total cell counts, and (F) neutrophils from BAL fluid of mice intratracheally treated with Lenti-Empty or Lenti-Itgb5 and then treated with LPS and PBS or recombinant MMP-9, as indicated. Data and means ± SEM pooled of 3 mice per group are from 2 independent experiments. (G) EBA uptake, (H) protein concentrations, (I) total cell counts, and (J) neutrophils from BAL fluid of mice intratracheally treated with Lenti-control shRNA or Lenti-Itgb5 shRNA and then treated with LPS and PBS or recombinant MMP-9, as indicated. Data and means ± SEM pooled of 3 mice per group are from 2 independent experiments. (K) Histological analysis of lung samples from mice treated as indicated. Images are representative of all independent experiments. (L) Survival rates of mice treated as indicated. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 5: MMP-9 regulates inflammation and permeability in LPS-treated cells. (A–D) PMVECs were treated with LPS (1µg/mL) with or without rMMP-9 (10 ng/mL) for 4 h. The mRNA levels of IL-6, CXCL-1, ICAM-1, and VCAM-1 were detected. (E) The protein levels of VCAM-1, ICAM-1 and TRAF-6 were detected by Western blot analysis in PMVECs exposed to LPS with or without rMMP-9 treatment. (F) MMP-9 protein levels were detected by immunoblotting in PMVECs exposed to LPS with or without PDTC treatment. (G) 293T cells were transfected with Cignal NF-kB dual luciferase reporter plasmids for 48 h, and then cells were treated with LPS (1µg/mL) with or without rMMP-9 (10 ng/mL) for an additional 6 h or 18 h. (H) 293T cells were co-transfected with NF-kB dual luciferase reporter plasmids along with control siRNA, or MMP-9 siRNA for 48 h, and then cells were treated with LPS (1µg/mL) for an additional 6 h or 18 h. Cells were then collected and assayed for luciferase activity to evaluate NF-kB promoter activity. (I) PMVECs were transfected with control siRNA, or MMP-9 siRNA for 48 h, and then cells were treated with LPS (1µg/mL) in a time course. Supernatant IL-6 secretion was measured by ELISA. (J) PMVECs were treated by APMA (APMA was used to active rMMP9 in vitro, and served as a control of active rMMP9) or active rMMP9 (10 ng/mL) 1 h before LPS challenged at indicated times, and then supernatant IL-6 was detected. (K and L) The permeability of treated PMVEC monolayers grown on 0.4 mm filters was determined by the appearance of rhodamine-dextran, which was added to the top well at the beginning of the experiment and detected the absorbance at 590 nm in the bottom well during a 90 min time course. *P<0.05, **P<0.01. All the results are from at least three independent experiments; Data represent means ±SEM.