Fig. 3: Defective response to glutamine under stress conditions in cilia-ablated cells.

a, Experimental design of LC–MS targeted metabolomics in MEF^Ctrl (CT) and MEF^Ift88 (KO) after 24 h culture in HBSS ± l-glutamine (Q) (4 mM). b, Volcano plot of metabolites in KO versus CT cultured in HBSS for 24 h as assessed by LC–MS targeted metabolomics as in a. FC, fold change. Black dots: P ≥ 0.05 and −0.5 ≤ log₂FC ≤ 0.5; grey dots: P ≥ 0.05 or −0.5 ≤ log₂FC ≤ 0.5; red dots: P < 0.05 and −0.5 > log₂FC > 0.5. c, Box-and-whisker plots of ATP/AMP, GTP/GMP and CTP/CMP ratio in MEF^Ctrl and MEF^Ift88 cells in HBSS for 24 h assessed by LC–MS targeted metabolomics as in a, n = 5 biological replicates. d, Top: Venn diagram showing the metabolites that significantly change in KO versus CT in HBSS and HBSS + Q. Light blue: 49 metabolites that change in KO versus CT only in HBSS. Rose: 14 metabolites that change in KO versus CT only in HBSS + Q. Purple: 37 metabolites that change in KO versus CT in HBSS and HBSS + Q. Bottom: Heat map showing the FC of HBSS + Q versus HBSS, in CT and KO, of the statistically different 51 metabolites. The 14 metabolites that change in KO versus CT in HBSS + Q are expressed in bold. Inf = Infinite. e, Left: analysis of OCR measurement of one representative experiment in MEF^Ctrl (CT) and MEF^Ift88 (KO) after 4 h culture in either HBSS (CT, blue; KO, green) or HBSS + l-glutamine (Q) (4 mM) (CT, red; KO, purple) in basal condition and after sequential addition of oligomycin (O), FCCP and antimycin A/rotenone (A/R). Right: quantification of basal respiration, ATP-production-coupled respiration and maximal respiration as in left. Box-and-whisker plots show median and minimum to maximum; data in bar plots are mean ± standard deviation. Statistical analysis: Student’s unpaired two-tailed t-test or one-way ANOVA, followed by Tukey’s multiple comparisons test; NS, not significant, ****P < 0.0001. n reported in brackets. Additional replicate experiment in e is shown in Supplementary Fig. 5.

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