Fig. 3: Ribosome impairment drives metabolic changes and the fetal signature via ZAKα activation.

a RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids treated with cycloheximide at either a low (0.015 ug/ml) or high dose (100 ug/ml) for 30 min. Hprt is used as a reference. Mean and SEM are shown (n = 3 biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test. b RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids cultured in media without glutamine or leucine for 1 h. Hprt is used as a reference. Mean and SEM are shown (n = 3 biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test. c Western blot confirming activation of ZAKα in Rptor^fl/fl and low-dose cycloheximide treated organoids (upper panel) and organoids deprived from glutamine and leucine (bottom panel) compared to WT. Red arrows indicate phosphorylated ZAKα, black arrows indicate the ZAKα levels, green stars indicate non-specific bands. Ponceau serves as a loading control. Blots were done on two animals. d RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids treated with low-dose cycloheximide (0.015 ug/ml) for 30 min in the presence or absence of ZAKα. Hprt is used as a reference. Mean and SEM are shown (n = 3 biological replicates each accessed in technical triplicates). p-values were determined using a two-tailed t-test. e OCR and ECAR analyses reveal that deleting ZAKα rescues the metabolic changes caused by low-dose cycloheximide (0.015 ug/ml). Mean and SD are shown (n = 1 biological replicate accessed in technical quadriplicates). f RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT and Rptor^fl/fl organoids in the presence or absence of ZAKα. Hprt is used as a reference. Mean and SEM are shown (n = 1 biological replicate accessed in technical triplicates). p-values were determined using a two-tailed t-test. g ECAR analysis show that ZAKα deletion rescues the metabolic changes caused by Rptor deletion. Mean and SD are shown (n = 1 biological replicate accessed in technical quadriplicates). h RT-qPCR analysis of genes related to stem (Lgr5) and fetal-like state (Tacstd2 and Sca1) of WT organoids grown in glutamine-depleted media, in the presence or absence of ZAKα. Hprt is used as a reference. Mean and SEM are shown (n = 1 biological replicate accessed in technical triplicates). p-values were determined using a two-tailed t-test.