Extended Data Fig. 6. AMPKα phosphorylates SMPD3 protein which became more stable through escaped from ubiquitination degradation.

a, Nicotine (1 μg/mL) treatment on Smpd3 mRNA levels in ileal organoids (n = 3/group). b, PRKAA1-WT or PRKAA1-KD (kinase domain mutant) was introduced into SW480 cells, and the cells were then treated with vehicle or nicotine (1 μg/mL) for 12 h. c, GPS2.0 predicts potential kinases and phosphorylation sites for SMPD3. d, The S208/209 peptide of SMPD3 satisfied the AMPK substrate motif and was conserved in different species (data from NCBI database). e, Mass spectrometry analysis of the phosphorylation at S209 on SMPD3. f, HFHCD-fed Prkaa1^fl/fl and Prkaa1^ΔIE mice (SPF) were treated with nicotine water for 2 weeks, and organoids were then isolated and cultured for 7 days and treated with nicotine (1 μg/mL) for the last 3 days. Western blot analysis showing the stability of SMPD3 after the administration of CHX. g, Mass spectrometry analysis of the ubiquitination at K103 on SMPD3. h, The K63 ubiquitination of SMPD3 in SW480 cells transfected with SMPD3-flag (WT and K103R) with or without nicotine treatment. i, The SMPD3 ubiquitination in SW480 cells transfected with SMPD3-flag (WT, S209A or S209A/K103R) and treated with or without nicotine. j, Phosphorylation level (S209) of SMPD3 was detected by anti-p-SPMD3 (S209) antibody in SW480 cells transfected with SMPD3-flag (WT or S209A) and treated with or without nicotine for 24 h. k, Ubiquitination level (K103) of SMPD3 was detected by anti-ubi-SMPD3 (K103) antibody in SW480 cells transfected with SMPD3-flag (WT or K103R) and treated with or without nicotine for 24 h. For e, g-k, nicotine (1 μg/mL) treatment for 24 h. Data are the means ± s.e.m. Experiments in a, b, e-k were performed three times independently. a, One-way ANOVA with Tukey’s post hoc test.