Extended Data Fig. 6: Referred to Fig. 4.
From: Primary cilia sense glutamine availability and respond via asparagine synthetase
a, Western blot for pS6RPS235/236 of total cell lysates from mIMCD3 cells after 24 hours culture in either DMEM/F12 ± 10% FBS or HBSS ± L-Glutamine (Q) (4 mM). Vinculin is used as loading control. b, Quantification of cilia length in one representative experiment in mIMCD3 cells after 24 hours culture in either HBSS ± Q or HBSS + Q + Rapamycin (100 nM). c, Western blot for pS6RPS235/236 of total cell lysates from mIMCD3 cells after 24 hours culture in either HBSS ± Q or L-Leucine (L) (0.5 mM). d, Quantification of cilia length in one representative experiment in mIMCD3 cells after 24 hours culture in either HBSS ± D-(+)-Glucose (Glc) (20 mM) or Q (4 mM) or L (5 mM). e, qRT-PCR analysis of Asns expression in one representative experiment in mIMCD3 transiently knocked down for Asns (siAsns) relative to control (Scr). f, Quantification of cilia length in one representative experiment in mIMCD3 after 8 hours culture in HBSS or HBSS + Q (4 mM). g, Quantification of cilia length in one representative experiment in mIMCD3 after 24 hours culture in either DMEM/F12 + 0% FBS or HBSS or HBSS + Q (0.5 or 4 mM) or HBSS + L-Asparagine (N) (0.1 or 1 mM). h, Left: analysis of OCR measurement in one representative experiment in siAsns MEF cells compared to Scr after 4 hours in HBSS (Scr: blue, siAsns: red) followed by acute injection (+) of Q (Scr: green, siAsns: purple), in basal condition and after sequential addition oligomycin (O), FCCP, and antimycin A/rotenone (A/R). Right: quantification of acute response as in Left. i, qRT-PCR analysis of Asns expression in one representative experiment in MEFIft88 and mIMCD3Ift88 relative to MEFCtrl and mIMCD3Ctrl. Data in dot and bar plots are mean ± SD. 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.
