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.

Effect of NC1, NC1-CUR, NC2 and NC2-CUR on SH-SY5Y cell viability. The cells were treated for 24 h with different dilutions(1:1, 1:2 and 1:4) of empty nanocapsules (NC1 and NC2) and nanocapsules containing curcumin (NC1-CUR and NC2-CUR) followed by measurement of cell viability by MTT reduction assay (panel A) and cell toxicity by LDH release assay (panel B). Data after normalization to vehicle-treated cells (100%, MTT assay) or to total LDH release (TritonX100-treated cells, 100%) are presented as a mean±SEM from 3-8 independent experiments with five replicates. *p < 0.05, **p < 0.01 and ***p<0.001 versus vehicle-treated cells; &&&p<0.001 NC1 versus NC1-CUR.

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

Treatment with triptolide selectively induces apoptosis of cultured and primary CD19+ CLL cells. A. Percent apoptosis (Annexin V-FITC and PI staining) in Mec-1, Mec-2 and WAC3-CD5+ cells exposed to the indicated doses of triptolide for 48 hrs; bars indicate standard deviation. B. Percent apoptosis of CD19+ normal and primary CLL (both high and low risk) cells exposed to the indicated doses of triptolide for 48 hrs; bars indicate standard deviation. C. Percent of WaC3-CD5+ and Mec-1 cells in different phases of the cell cycle (G0/G1, S and G2/M) exposed to the indicated doses of triptolide for 24 hrs. D. Immunoblot analyses of HSF1, HSP70, cleaved caspase-3 and β-actin obtained from the cell lysates of CD19+ primary CLL cell samples treated with the indicated doses of triptolide for 24 hrs.

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.

Effect of NC1, NC1-CUR, NC2 and NC2-CUR on SH-SY5Y cell viability. The cells were treated for 24 h with different dilutions(1:1, 1:2 and 1:4) of empty nanocapsules (NC1 and NC2) and nanocapsules containing curcumin (NC1-CUR and NC2-CUR) followed by measurement of cell viability by MTT reduction assay (panel A) and cell toxicity by LDH release assay (panel B). Data after normalization to vehicle-treated cells (100%, MTT assay) or to total LDH release (TritonX100-treated cells, 100%) are presented as a mean±SEM from 3-8 independent experiments with five replicates. *p < 0.05, **p < 0.01 and ***p<0.001 versus vehicle-treated cells; &&&p<0.001 NC1 versus NC1-CUR.

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

Treatment with triptolide selectively induces apoptosis of cultured and primary CD19+ CLL cells. A. Percent apoptosis (Annexin V-FITC and PI staining) in Mec-1, Mec-2 and WAC3-CD5+ cells exposed to the indicated doses of triptolide for 48 hrs; bars indicate standard deviation. B. Percent apoptosis of CD19+ normal and primary CLL (both high and low risk) cells exposed to the indicated doses of triptolide for 48 hrs; bars indicate standard deviation. C. Percent of WaC3-CD5+ and Mec-1 cells in different phases of the cell cycle (G0/G1, S and G2/M) exposed to the indicated doses of triptolide for 24 hrs. D. Immunoblot analyses of HSF1, HSP70, cleaved caspase-3 and β-actin obtained from the cell lysates of CD19+ primary CLL cell samples treated with the indicated doses of triptolide for 24 hrs.