Extended Data Fig. 5: Effects of the UPR and ISR on LIF release.
A. Relative expression of indicated mRNA, measured by qPCR, after 24 h of treatment (n = 3). B. Relative expression of indicated mRNA, measured by qPCR, after silencing (48 h) and 24 h of further treatment (n = 3). C. Relative expression of ATF6, measured by qPCR, after silencing (48 h) and 16 h of further treatment (n = 3). D. Western blot of ATF6 after silencing (48 h) and subsequent treatment (16 h). E. LIF secretion in the cell lines indicated after siRNA silencing for 48 h with ATF6. Incubated with glucose-free media for 16 h (n = 3 for A549, 5 for H1299 and SW900). F-H. Protein content per well measured by BCA of panel Fig. 5e-f (F,H n = 3, G n = 4). I. LIF in supernatants of A549 cells subject to PERK knockdown (24 h) and subsequent glucose deprivation (24 h), normalized to protein content (n = 4). J. Protein content per well measured by BCA of panel I (n = 4). K. Western blot of PERK after silencing (24 h) and subsequent treatment (24 h) in A549 cells. L. Western blot of LLC cells treated with glucose deprivation and the ISR inhibitor ISRIB (6 h) in media with 10% dFBS. M. Secreted LIF in supernatants of LLC cells under glucose deprivation (6-16 h) and treated with ISRIB, normalized to protein content (n = 3). N. Protein content per well (panel 5H, n = 3). O. Western blot of ATF4 after silencing (48 h) and subsequent treatment (16 h). P. As in E, with siRNA against ATF4 (n = 6 for A549, 4 for H1299 and SW900). Q. Protein content per well (Fig. 5i and Extended Data Fig. 5P, H1299, n = 3) R. Cells treated with non-targeting siRNA (“c”) or siRNA against QRICH were treated for 16 h and subjected to WB (up) and their supernatant to LIF ELISA (bottom, n = 3). S. Protein content per well (panel 5 K, n = 3) T. Cells treated with thapsigargin at the indicated concentrations for 24 h and subjected to WB (up), and their supernatants to LIF ELISA (bottom, n = 3). U-V. LIF secretion (non-glycosylated in U, n = 4, or total in V, n = 1) from A549 24 h after silencing p65 for 48 h. W. Protein content per well (panel 5 M, n = 3). X. Densitometric analysis of phospho ERK1/2 normalized to 1 h glc+ (Fig. 5o, n indicated in figure). Graphs show average values and individual replicates, and error bars represent the standard error of the mean (SEM); statistical significance was determined by one-way ANOVA with multiple comparisons test (A, B, C and Q), two-way ANOVA (F, G, M, N, S, W, X) or two-tailed paired t-test (E, H, I, J, P, R, U, X).
