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. Author manuscript; available in PMC: 2019 Jun 17.
Published in final edited form as: Stem Cell Res. 2019 Mar 27;37:101430. doi: 10.1016/j.scr.2019.101430

Fig. 2.

Fig. 2

L-WRN CM activity is maintained over several weeks of storage following thaw.

(A-E) L-WRN CM was stored at 4C for 0WK, 1WK, 2WK, 3WK or subjected to a second freeze-thaw cycle (2XFT). An aliquot of the 2XFT sample was removed prior to the second freeze-thaw cycle to serve as a direct control (2XFT Cont). Spheroids cultured in differentiation medium with EP4 inhibitor (DM + EP4i), treated with cycloheximide and tumor necrosis factor (CHX + TNF), or treated with butyrate served as negative controls. (A) Schematic of experimental time line for assays in (B) and (C). (B) Graph of CellTiter-Glo data presented as fold change (mean ± s.e.m.) relative to DM + EP4i; n = 3 independent experiments. ****P < 0.0001 by 1-way ANOVA and Dunnett’s post test relative to 0WK. (C) Graphs of mRNA gene expression for indicated genes as determined by qPCR. Data are presented as fold change (mean ± s.e.m.) relative to 0WK; n = 3 independent experiments. (D) Schematic of experimental time line for (E). (E) Graph of Cdc25A-CBRluc data normalized to the average 0 h value of all samples; n = 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 1-way ANOVA (B, C) or by 2-way repeated measures ANOVA (E) using Dunnett’s post test relative to 0WK (B, C, E).