Extended Data Fig. 2: Kinetics of secretion of glycosylated and unglycosylated LIF.
A. LIF secretion kinetics measured using R&D hLIF Quantikine ELISA (total LIF, upper panels n ≥ 3, check figure for exact n values) and R&D hLIF Duoset ELISA (for non-glycosylated LIF, lower panels, n = 3). Glc + in grey and Glc 0 mM in red. B. LIF mRNA kinetics in cellular lysates from A549 cells incubated in Plasmax measured by qPCR (n = 4 (0-24 h), 3(48 h), 2(72 h)). C. Translation efficiency of LIF, calculated as the ratio between ribosome protected fragments and RNA expression in an immortalized breast epithelial cell line (MCF10A) untransformed (EtOH) and transformed (TAM) to tumorigenic breast epithelium, and subject to glucose or glutamine deprivation for 30 min and 4 h. Calculated from public data, n = 2 (Gameiro & Struhl, 2018)7. D. LIF protein secretion, not normalized to protein content, in A549 cells after incubation for 24 h in 0 or 25 mM Glc (grey or red) in presence or absence of actinomycin D (ActD) (n = 3). E. LIF protein secretion in SW900 cells as in D, after incubation for 16 h (n = 4 (total LIF), n = 3 (non-glyc LIF)). F. LIF protein measured using R&D hLIF Quantikine ELISA (total LIF) in supernatants from A549 cells after 24 h incubation in presence or absence of glucose and increasing concentrations of H2O2. Results normalized by protein content (n = 3). G. Secreted LIF protein from A549 cells after 24 h treatment with the inhibitor of glucose-6-phosphate dehydrogenase, dehydroepiandrosterone (DHEA), at indicated concentrations, measured using R&D hLIF Quantikine ELISA (total LIF). Results normalized to protein content (n = 2). H. Protein content per well after incubating cells for 24 h under hypoxic conditions (0.1 or 1% O2) in DMEM + 10% dFBS (A549 (n = 3), H1299 (n = 4) and LLC (n = 5)) or RPMI + 10% dFBS (SW900, n = 3). I. LIF protein measured using ELISA in supernatants from A549 cells after 24 h incubation in presence or absence of glucose and increasing concentrations of CoCl2. Results were normalized by protein content (n = 3). J. LIF mRNA expression measured by qPCR and expressed as relative expression versus normoxia conditions (21% O2) after incubating cells for 24 h under hypoxic conditions (1% O2) in DMEM + 10% dFBS (H1299 and LLC, n = 3) or RPMI + 10% dFBS (SW900, n = 3). Graphs show average values and individual replicates, and error bars represent the standard error of the mean (SEM); statistical significance was determined by two-way ANOVA with multiple comparisons test (A-B), one-way ANOVA with multiple comparisons test (D-G and I) or two-tailed paired t-test (H and J).
