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2.
Figure 6

Figure 6. Mechanism for EF31-dependent inhibition of the NF-κB pathway. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

IκKβ recombinant protein was pre-incubated with EF24, EF31 (0.0977, 0.391, 1.56, 6.25, 25, 100 µM), or vehicle (DMSO 1%) for 30 minutes. Inhibition of IκKβ activity was measured using a Z’-Lyte kinase assay kit as described in section 2.7. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. ** p < 0.01, *** p < 0.001 vs. EF24 at same concentration.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
3.
Figure 9

Figure 9. EF31 shows potent toxicity on NF-κB dependent cancer cell lines. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Cancer cell lines were grown in 96-well plates and treated with curcumin, EF31 (1, 5, 10, or 50 µM), or vehicle (DMSO 1%) for 48 hours. Cell viability/ proliferation was measured as described in section 2.8. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent of vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. curcumin at same concentration.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
4.
Figure 11

Figure 11. EF31 inhibits lipopolysaccharide-induced phosphorylation of MAPKs p38, JNK, and ERK. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pre-treated with EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 or 30 minutes. Whole cell extracts and western blot analyses were conducted for unphosphorylated and phosphorylated (p) p38 (A), JNK (B), and ERK (C) and as described in section 2.9. Results shown are representative of three separate experiments.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
5.
Figure 7

Figure 7. Inhibition of NF-κB DNA-binding by EF31 is reversible. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pre-treated with curcumin (50 µM), EF24(50 µM), EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour. Cells were then washed and medium was replaced and treated with LPS (1 µg/mL) or saline. Nuclear proteins were collected at different time-points (15 minutes– 6 hours) as described in section 2.3. NF-κB DNA-binding activity was measured using a DNA-binding ELISA. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM). * p < 0.05 vs. vehicle + LPS at 15 minutes.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
6.
Figure 10

Figure 10. EF31 inhibits MAPK transcription factor DNA-binding activity. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pretreated with curcumin (50 µM), EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 minutes. Nuclear proteins were collected as described in section 2.3 and transcription factor DNA-binding was measured using a DNA-binding ELISA for ATF-2 (top) and c-Jun (bottom). Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM). *** p < 0.001 vs. curcumin + LPS; ††† p < 0.001 vs. vehicle + LPS; aa p < 0.01, aaa p < 0.001 vs. vehicle + saline.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
7.
Figure 8

Figure 8. EF31 shows no reduction in cell viability at concentrations that inhibit NF-κB. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Mouse RAW264.7 macrophages were grown in 96-well plates and pre-treated with curcumin, EF24, EF31 (1, 5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 minutes. Cell viability/ proliferation was then measured as described in section 2.8. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent of vehicle. ** p < 0.01, *** p < 0.001 vs. curcumin at same concentration in the saline treated groups; a p < 0.05 vs. EF24 at same concentration in the saline treated groups; †† p < 0.01 vs. curcumin at same concentration in the LPS treated groups; bb p < 0.01 vs. EF24 at same concentration in the LPS treated groups.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
8.
Figure 4

Figure 4. EF31 inhibits LPS-induced pro-inflammatory cytokine mRNA expression. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Mouse RAW264.7 macrophages were grown in 6-well plates and pre-treated with curcumin, EF24, EF31 (5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 3 hours. Whole cell mRNA was then collected as described in section 2.5 to assess TNF-alpha (A), IL-1 beta (B), and IL-6 (C) gene expression using RT-PCR. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. *** p < 0.001 vs. curcumin at same concentration; † p < 0.05, †† p < 0.01, ††† p < 0.001 vs. EF24 at same concentration.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
9.
Figure 5

Figure 5. EF31 inhibits LPS-induced production of pro-inflammatory cytokine proteins. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

Mouse RAW264.7 macrophages were grown in 6-well plates and pre-treated with curcumin, EF24, EF31 (5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 16 hours. Medium was collected and assessed for concentrations of TNF-alpha (A), IL-1 beta (B), and IL-6 (C) protein using ELISAs. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. curcumin at same concentration; †† p < 0.01, ††† p < 0.001 vs. EF24 at same concentration. (Of note, LPS + DMSO treated samples yielded protein levels of approximately 1,400 pg/mL for TNF-α, 15 pg/mL for IL-1β, and 11,000 pg/mL for IL-6)

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
10.
Figure 3

Figure 3. EF31 impairs LPS-induced NF- κB nuclear translocation. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

A) Mouse RAW264.7 macrophages were grown in 8-well chamber slides and pre-treated with curcumin, EF24, EF31 (5, 10, or 50 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 15 minutes. Cells were then fixed and processed as described in section 2.4. Images were obtained using a LSM510 confocal microscope. Scale bar = 5µm. B) The induction of NF-κB nuclear translocation by LPS was quantified by measuring nuclear p65 fluorescence intensity as described in section 2.4. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. *** p < 0.001 vs. curcumin at same concentration; †† p < 0.01 vs. EF24 at same concentration; a p < 0.001 vs. vehicle.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.
11.
Figure 2

Figure 2. EF31 is a potent inhibitor of LPS-induced NF-κB DNA binding activity. From: Inhibition of the NF-κB signaling pathway by the curcumin analog, 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties.

A) Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and treated with LPS (1 or 10 µg/mL) or saline. Nuclear proteins were collected at different time-points (5 minutes– 60 minutes) as described in section 2.3. NF-κB DNA-binding activity was measured using a DNA-binding ELISA. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM). * p < 0.05 vs. 5 minutes- LPS (1 µg/mL) treated group. B) Mouse RAW264.7 macrophages were pre-treated with curcumin, EF24, EF31 (1, 5, 10, 30, 50, or 100 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) for 15 minutes. Nuclear proteins were collected and NF-κB DNA-binding was measured using a DNA-binding ELISA. Results shown are representative of three separate experiments. All conditions were run in triplicate, and values shown are means (±SEM) expressed as percent inhibition from vehicle. * p < 0.05, *** p < 0.001 vs. curcumin at same concentration; †† p < 0.01, ††† p < 0.001 vs. EF24 at same concentration. C) Mouse RAW264.7 macrophages were grown in 60 mm × 15 mm cell culture dishes and pre-treated with EF31 (10 µM), or vehicle (DMSO 1%) for 1 hour prior to treatment with LPS (1 µg/mL) or saline for 15 or 30 minutes. Whole cell extracts and western blot analyses were conducted for unphosphorylated and phosphorylated IkB and NFkB p65 as described in section 2.9. Results shown are representative of three separate experiments.

Anlys Olivera, et al. Int Immunopharmacol. ;12(2):368-377.

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