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. Author manuscript; available in PMC: 2022 Jan 16.
Published in final edited form as: Cell Rep. 2021 Nov 16;37(7):110013. doi: 10.1016/j.celrep.2021.110013

Figure 3. Lysolipid species and secreted lysoPLD activity in conditioned media from melanoma cells.

Figure 3.

(A) Preparation of cell-conditioned media. Melanoma cells in 10-cm dishes were cultured for 24 h, washed, and then incubated in DMEM containing 0.5% FCS. Media were harvested after 24 and 48 h, and centrifuged to remove cell debris. LPA species were measured using LC/MS/MS.

(B) Determination of LPA species in conditioned medium from MDA-MB-435 and A375 melanoma cells, measured at t = 0, 24, and 48 h, using LC/MS/MS. Predominant serum-borne LPA species are (12:0), (16:0), (18:0), (18:1) and (20:4). Note LPA depletion from the medium (within 24 h) upon incubation with ATX-secreting melanoma cells.

(C) Time-dependent decline of the indicated serum-borne LPA species by melanoma cells. Graph shows normalized steady-state LPA levels in conditioned media from MDA-MB-435 cells.

(D) LPC species in conditioned medium from MDA-MB-435 cells, measured at t = 10 min, 2 h and 24 h, using LC/MS/MS. Note that LPC levels tend to increase over time. Values from one experiment performed in triplicate and expressed as mean ± SEM.

(E) Secreted lysoPLD activity increases over time. Medium from MDA-MB-435 cells was collected after 2 and 24 h, and lysoPLD activity was measured as choline release from added LPC(18:1). Values from three independent experiments each performed in triplicate and expressed as mean ± SEM; **p < 0.01 (unpaired Student’s t test).