HaCaT Eicosanoid Analysis Results

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Calcium Ionophore

AA-derived lipid mediators detected in the supernatant of HaCaT keratinocytes treated with A23187 (5 µM), for 0.5, 1, 3 and 6h. Analysis was performed using UPLC-ESI-MS/MS. Results are expressed mean ± SD (pg/106 cells) of three independent experiments.

Time (h)	Mediator
	PGE2	15-keto-PGE2	13,14-dihydro-15-keto-PGE2	PGF2α	12-HETE	15-HETE
0	9.45 ± 7.56	0.50 ± 0.00	2.00 ± 0.00	7.00 ± 0.00	6.44 ± 4.40	3.72 ± 1.08
0.5	601.26 ± 162.02	65.52 ± 57.95	211.38 ± 61.05	106.06 ± 39.89	19.49 ± 10.36	163.26 ± 143.10
1	14371.34 ± 858.76	2136.87 ± 588.92	2461.84 ± 452.79	2787.86 ± 489.04	363.25 ± 96.91	4050.14 ± 2998.16
3	79576.88 ± 15355.19	12230.83 ± 2179.66	11025.93 ± 2287.76	19276.46 ± 3732.43	4872.78 ± 849.95	41799.69 ± 31552.30
6	111115.14 ± 32976.86	18619.32 ± 5691.99	15799.31 ± 5867.25	34965.99 ± 11250.10	7156.92 ± 1550.86	55738.98 ± 40130.05

Lipid mediator levels in unstimulated (red) and stimulated with A23187 (5 µM) (blue) HaCaT keratinocytes over 6h. Where (A) PGE2, (B) 15-keto-PGE2, (C) 13,14-dihydro-15-keto-PGE2, (D) PGF2α, (E) 12-HETE and (D) 15-HETE. The data presented is from three independent experiments, each with a technical replicate.

ATP

AA-derived lipid mediators detected in the supernatant of HaCaT keratinocytes treated with ATP (2 mM), for 0.5, 1, 3 and 6h. Analysis was performed using UPLC-ESI-MS/MS. Results are expressed mean ± SD (pg/106 cells) of three independent experiments.

Time (h)	Mediator
	PGE2	15-keto-PGE2	13,14-dihydro-15-keto-PGE2	PGF2α	12-HETE	15-HETE
0	1.50 ± 0.00	0.37 ± 0.21	2.50 ± 0.00	7.50 ± 0.00	1.00 ± 0.00	2.00 ± 0.00
0.5	617.77 ± 60.96	1.10 ± 0.58	51.51 ± 6.47	96.88 ± 13.81	49.56 ± 37.94	15.82 ± 9.10
1	849.12 ± 56.72	3.60 ± 0.79	86.88 ± 32.86	148.10 ± 20.14	32.28 ± 31.72	10.95 ± 9.46
3	833.49 ± 170.07	2.05 ± 0.65	106.43 ± 12.88	170.02 ± 34.73	34.05 ± 39.23	5.70 ± 7.02
6	1015.73 ± 88.65	2.43 ± 1.70	141.16 ± 9.62	210.30 ± 43.78	9.76 ± 10.52	2.22 ± 0.54

Lipid mediator levels in unstimulated (red) and stimulated with ATP (2mM) (blue) HaCaT keratinocytes over 6h. Where (A) PGE2, (B) 15-keto-PGE2, (C) 13,14-dihydro-15-keto-PGE2, (D) PGF2α, (E) 12-HETE and (D) 15-HETE. The data presented is from three independent experiments, each with a technical replicate.

UVR

AA-derived lipid mediators detected in the supernatant of HaCaT keratinocytes treated with UVR (15 mJ/cm2), for 0.5, 1, 3 and 6h. Analysis was performed using UPLC-ESI-MS/MS. Results are expressed mean ± SD (pg/106 cells) of three independent experiments.

Time (h)	Mediator
	PGE2	15-keto-PGE2	13,14-dihydro-15-keto-PGE2	PGF2α	12-HETE	15-HETE
0	203.29 ± 315.50	0.50 ± 0.00	2.00 ± 0.00	0.00 ± 0.00	3.50 ± 0.00	2.30 ± 0.00
0.5	1.50 ± 0.00	0.75 ± 0.39	2.00 ± 0.00	88.72 ± 33.06	135.07 ± 142.32	40.09 ± 35.24
1	69.26 ± 60.99	1.05 ± 0.99	2.29 ± 1.56	90.56 ± 11.96	131.60 ± 100.39	46.54 ± 35.33
3	1.50 ± 0.00	9.61 ± 7.15	3.53 ± 2.41	222.44 ± 6.73	103.71 ± 78.12	58.45 ± 43.57
6	5667.91 ± 4967.29	152.58 ± 51.19	6.21 ± 4.30	1657.38 ± 297.77	766.69 ± 206.41	648.45 ± 219.84

Lipid mediator levels in unstimulated (red) and irradiated (15 mJ/cm2) (blue) HaCaT keratinocytes over 6h. Where (A) PGE2, (B) 15-keto-PGE2, (C) 13,14-dihydro-15-keto-PGE2, (D) PGF2α, (E) 12-HETE and (D) 15-HETE. The data presented is from three independent experiments, each with a technical replicate.

Calcium ionophore + Indomethacin

AA-derived lipid mediators detected in the supernatant of COX inhibited HaCaT keratinocytes treated with A23187 (5 µM), for 0.5, 1, 3 and 6h. Analysis was performed using UPLC-ESI-MS/MS. Results are expressed mean ± SD (pg/106 cells) of three independent experiments.

Time (h)	Mediator
	PGE2	15-keto-PGE2	13,14-dihydro-15-keto-PGE2	PGF2α	12-HETE	15-HETE
0	26.81 ± 10.56	0.50 ± 0.00	2.00 ± 0.00	7.00 ± 0.00	0.29 ± 0.00	1.92 ± 0.94
0.5	34.23 ± 22.16	0.50 ± 0.00	1.88 ± 0.57	7.00 ± 0.00	7.20 ± 10.94	29.71 ± 60.25
1	25.85 ± 9.25	0.50 ± 0.00	32.91 ± 28.56	7.00 ± 0.00	141.09 ± 24.66	54.23 ± 51.50
3	4731.85 ± 7300.78	2266.13 ± 371.89	1817.62 ± 907.18	3268.46 ± 658.45	17918.03 ± 8595.61	55405.98 ± 17910.03
6	4843.70 ± 7474.03	1620.01 ± 317.98	900.50 ± 199.18	4754.66 ± 596.47	61471.62 ± 16768.21	105543.44 ± 41314.43

Lipid mediator levels in unstimulated (red) and stimulated with A23187 (5 µM) following 1h incubation with indomethacin (10 mM) (blue) HaCaT keratinocytes, over 6h. Where (A) PGE2, (B) 15-keto-PGE2, (C) 13,14-dihydro-15-keto-PGE2, (D) PGF2α, (E) 12-HETE and (D) 15-HETE. The data presented is from three independent experiments, each with a technical replicate.