HaCaT Eicosanoid Analysis Results

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The aim of this study was to investigate the levels of AA derived lipid mediators in the supernatant following stimulation of cells with A23187 (with and without COX inhibition), ATP and UVR. LC-MS/MS was used for the eicosanoid analysis and the values were normalized using cell numbers (pg/10^6 cells).

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.