NF-κB

TE7 cells (2 × 10⁵) were pretreated with PD98059 (20 μM), QNZ (EVP4593; 20 μM), AZD5363 (20 μM), S1155 (20 μM) and Tra (40 ng/ml) for 1 h and stimulated with IL6 (40 ng/ml) for 48 h.

A. Raw264.7 cells were pretreated with NAC (1 mM), SB202190 (10 μM) and Sodium 4-aminosalicylate (10 μM), respectively, for 30 min prior to 10 μg/ml ConA treatment for 24 h. Cell viability was measured by an MTT assay and ROS production detected by H2DCF-DA.

CD10+GPR77+ CAFs were transduced without (-) or with GFP-shRNA or p65 shRNA or pretreated with inhibitors of NF-kB nuclear translocation. Quantitation of the percentage of CD10+GPR77+ CAFs (D) and the IL-6 and IL-8 levels (E) were shown.

The protein expression alteration of p-NF-κB, NF-κB, PD-L1 in NP-69-LMP1 or NP-69 cell lines treated with 0, 0.5, and 1.0 μM Caffeic Acid Phenethyl Ester (CAPE) for 72 hours. β-actin was used to verify equal loading.

Evodiamine ameliorates PDGF‑BB‑induced oxidative stress in VSMCs. VSMCs were pretreated with 0.1 or 0.5 μM evodiamine for 24 h, and then stimulated with 10 ng/ml PDGF‑BB for 1 h. (A) ROS detection using 2' 7' dichlorofluorescin diacetate staining. Magnification, x200. (B) Quantitative data of five independent experiments, expressed as the fold increase, compared with the control. (C‑F) mRNA expression levels of antioxidant genes were assessed using reverse transcription‑quantitative polymerase chain reaction analysis. Data are presented as the mean+ standard deviation of three independent experiments. *P<0.05 and **P<0.01, compared with the control group; ##P<0.01, compared with the PDGF‑BB‑stimulated group. Evo, evodiamine; PDGF‑BB, platelet‑derived growth factor‑BB; VSMCs, vascular smooth muscle cells; ROS, reactive oxygen species; HO‑1, heme oxygenase‑1; GPx‑1, glutathione peroxidase 1; SOD, superoxide dismutase.

CON+ -positive control (with FBS as chemoattractant); CON- - negative control (without chemoattractant); DOX -doxorubicin; WZ -WZ811; PF- PF-573228; * -significant difference compared to positive control: p<0.05; ** -significant difference compared to positive control: p<0.01; # -significant difference between combination and inhibitor only: p<0.05. Cancer cell invasion was analyzed using Transwell membranes assembled in 24-well plates and covered with a layer of Matrigel. COR-L23 cells were treated for 24 h with 50 nM DOX, 10 uM WZ811, 5 uM PF-573228 and combinations of inhibitors with 50 nM DOX. Similarly, RH460 cells were treated for 24 h with 1 uM DOX, 10 uM WZ811, 2.5 uM PF-573228 and combinations of inhibitors with 1 uM DOX. Applied doses were the ones that exerted the most pronounced synergistic effect on cytotoxicity level. After the incubation period, cells that invaded through matrigel and membranes were fixed in 4% paraformaldehyde-PBS and stained with Hoechst 33342 nuclear stain. Cells were visualized and counted under a fluorescent microscope at 10x magnification.

Immunofluorescence of NF-κB (p65) (200×). SK-OV-3 and A2780S cells were treated with different compounds (vehicle, 5 μM JSH-23, 5 μM SC75741, 5 μM CAPE, 4 μM MIL, 4 μM MIL combined with pre-treated 5 μM JSH-23, 4 μM MIL combined with pre-treated 5 μM SC75741, 4 μM MIL combined with pre-treated 5 μM CAPE) for 48 h. NF-κB(p65) was detected by a fluorescence microscopy. The nuclei were stained with Hoechst 33258.

Magnolol reduces RNV areas and avascular areas in OIR mice. Mice retinas from OIR group (a, b) and OIR + Magnolol (25 mg/kg) group (c, d) were harvested at P17 and subjected to wholemount immunostaining with IB4 (red), showing neovascular areas (green) and avascular areas (yellow).

 G. To evaluate effects of IKBKE/TBK1 inhibition on NF-κB signaling in Ewing, TC32 cells were incubated with CYT387 for six hours prior to stimulation with TNF-α (30 ng/mL). IκBα degradation was measured by harvesting TC32 cells thirty minutes after stimulation with TNF-α. TNF-α stimulation resulted in degradation of IκBα, and this effect was attenuated with CYT387 treatment. Parthenolide, an inhibitor of IκBα phosphorylation was used as a positive control. Similar effects of CYT387 activity were seen in HEK-293T cells which also express IKBKΕ. Nuclear extracts were prepared from TC32 cells harvested following forty-five minutes of TNF-α stimulation. Treatment with CYT387 resulted in decreased nuclear localization of NF-κB family proteins RelA/p65 and c-Rel. There was a modest impairment of p50 nuclear localization as compared to parthenolide and DMSO controls and no change in p52 nuclear localization. RelB (not shown) is not expressed in TC32 cells

Spironolactone (sp.) and triptolide inhibit NER in myeloma cells. RPMI8226 cells were incubated with dimethyl sulfoxide (DMSO), spironolactone (10 μm) or triptolide (1 μm) for 6 h before NER evaluation. The figure represents the persistence of DNA-damage signal 150 min after exposure to UV (AFU: arbitrary fluorescent unit). Figure 4a shows representative merged pictures of DAPI and DDB2 proteo-probe signal (b).

TE7 cells (2 × 10⁵) were pretreated with PD98059 (20 μM), QNZ (EVP4593; 20 μM), AZD5363 (20 μM), S1155 (20 μM) and Tra (40 ng/ml) for 1 h and stimulated with IL6 (40 ng/ml) for 48 h.

A. Raw264.7 cells were pretreated with NAC (1 mM), SB202190 (10 μM) and Sodium 4-aminosalicylate (10 μM), respectively, for 30 min prior to 10 μg/ml ConA treatment for 24 h. Cell viability was measured by an MTT assay and ROS production detected by H2DCF-DA.

CD10+GPR77+ CAFs were transduced without (-) or with GFP-shRNA or p65 shRNA or pretreated with inhibitors of NF-kB nuclear translocation. Quantitation of the percentage of CD10+GPR77+ CAFs (D) and the IL-6 and IL-8 levels (E) were shown.

The protein expression alteration of p-NF-κB, NF-κB, PD-L1 in NP-69-LMP1 or NP-69 cell lines treated with 0, 0.5, and 1.0 μM Caffeic Acid Phenethyl Ester (CAPE) for 72 hours. β-actin was used to verify equal loading.

Evodiamine ameliorates PDGF‑BB‑induced oxidative stress in VSMCs. VSMCs were pretreated with 0.1 or 0.5 μM evodiamine for 24 h, and then stimulated with 10 ng/ml PDGF‑BB for 1 h. (A) ROS detection using 2' 7' dichlorofluorescin diacetate staining. Magnification, x200. (B) Quantitative data of five independent experiments, expressed as the fold increase, compared with the control. (C‑F) mRNA expression levels of antioxidant genes were assessed using reverse transcription‑quantitative polymerase chain reaction analysis. Data are presented as the mean+ standard deviation of three independent experiments. *P<0.05 and **P<0.01, compared with the control group; ##P<0.01, compared with the PDGF‑BB‑stimulated group. Evo, evodiamine; PDGF‑BB, platelet‑derived growth factor‑BB; VSMCs, vascular smooth muscle cells; ROS, reactive oxygen species; HO‑1, heme oxygenase‑1; GPx‑1, glutathione peroxidase 1; SOD, superoxide dismutase.

CON+ -positive control (with FBS as chemoattractant); CON- - negative control (without chemoattractant); DOX -doxorubicin; WZ -WZ811; PF- PF-573228; * -significant difference compared to positive control: p<0.05; ** -significant difference compared to positive control: p<0.01; # -significant difference between combination and inhibitor only: p<0.05. Cancer cell invasion was analyzed using Transwell membranes assembled in 24-well plates and covered with a layer of Matrigel. COR-L23 cells were treated for 24 h with 50 nM DOX, 10 uM WZ811, 5 uM PF-573228 and combinations of inhibitors with 50 nM DOX. Similarly, RH460 cells were treated for 24 h with 1 uM DOX, 10 uM WZ811, 2.5 uM PF-573228 and combinations of inhibitors with 1 uM DOX. Applied doses were the ones that exerted the most pronounced synergistic effect on cytotoxicity level. After the incubation period, cells that invaded through matrigel and membranes were fixed in 4% paraformaldehyde-PBS and stained with Hoechst 33342 nuclear stain. Cells were visualized and counted under a fluorescent microscope at 10x magnification.

Immunofluorescence of NF-κB (p65) (200×). SK-OV-3 and A2780S cells were treated with different compounds (vehicle, 5 μM JSH-23, 5 μM SC75741, 5 μM CAPE, 4 μM MIL, 4 μM MIL combined with pre-treated 5 μM JSH-23, 4 μM MIL combined with pre-treated 5 μM SC75741, 4 μM MIL combined with pre-treated 5 μM CAPE) for 48 h. NF-κB(p65) was detected by a fluorescence microscopy. The nuclei were stained with Hoechst 33258.

Magnolol reduces RNV areas and avascular areas in OIR mice. Mice retinas from OIR group (a, b) and OIR + Magnolol (25 mg/kg) group (c, d) were harvested at P17 and subjected to wholemount immunostaining with IB4 (red), showing neovascular areas (green) and avascular areas (yellow).

 G. To evaluate effects of IKBKE/TBK1 inhibition on NF-κB signaling in Ewing, TC32 cells were incubated with CYT387 for six hours prior to stimulation with TNF-α (30 ng/mL). IκBα degradation was measured by harvesting TC32 cells thirty minutes after stimulation with TNF-α. TNF-α stimulation resulted in degradation of IκBα, and this effect was attenuated with CYT387 treatment. Parthenolide, an inhibitor of IκBα phosphorylation was used as a positive control. Similar effects of CYT387 activity were seen in HEK-293T cells which also express IKBKΕ. Nuclear extracts were prepared from TC32 cells harvested following forty-five minutes of TNF-α stimulation. Treatment with CYT387 resulted in decreased nuclear localization of NF-κB family proteins RelA/p65 and c-Rel. There was a modest impairment of p50 nuclear localization as compared to parthenolide and DMSO controls and no change in p52 nuclear localization. RelB (not shown) is not expressed in TC32 cells

Spironolactone (sp.) and triptolide inhibit NER in myeloma cells. RPMI8226 cells were incubated with dimethyl sulfoxide (DMSO), spironolactone (10 μm) or triptolide (1 μm) for 6 h before NER evaluation. The figure represents the persistence of DNA-damage signal 150 min after exposure to UV (AFU: arbitrary fluorescent unit). Figure 4a shows representative merged pictures of DAPI and DDB2 proteo-probe signal (b).