Fig. 3: PANK4 suppresses CoA synthesis and phosphorylation of PANK4 Thr406 reduces this suppression.
From: PI3K drives the de novo synthesis of coenzyme A from vitamin B5
a, PANK kinase inhibitor (0–10 μM; inhib.) treatments with concurrent 13C315N1-VB5 labelling (3 h) of MCF10A cells expressing doxycycline-inducible HA-tagged wild-type or constitutively active (E17K) AKT. Treatment and labelling were preceded by doxycycline incubation (48 h), serum and growth factor deprivation (18 h) and inhibitor pretreatment (15 min). b, Individual siRNA-mediated knockdowns of PANK1, PANK2 and PANK4 (siP1, siP2 and siP4, respectively) or non-targeting siRNA (siC) in MCF10A AKTp.E17K/+ cells with 13C315N1-VB5 labelling (24 h) preceded by serum and growth factor deprivation (18 h). c, Wild-type PANK4 and PANK4-KO AKTp.E17K/+ MCF10A cells with 13C315N1-VB5 labelling (3 h) preceded by serum and growth factor deprivation (18 h). Divided blots are from same SDS–PAGE gel and image. d, PANK4-KO cells stably expressing vector (Vec) or untagged human PANK4 (WT or T406A) with conditions and labelling otherwise as described in c. Divided blots are from same SDS–PAGE gel and image. e, ACLY inhibitor (NDI-091143, 20 μM) treatment with concurrent 13C315N1-VB5 labelling (3 h) of PANK4-KO cells stably expressing vector or untagged human PANK4 (T406A or T406E). Treatments and labelling were preceded by serum and growth factor deprivation (18 h) and inhibitor pretreatment (2 h). f, Two-dimensional (2D) proliferation of cell lines from d and e with growth factors. Statistical comparison was performed on day 4 data. For a–e, metabolites were measured using LC–MS/MS and normalized to protein; labelled metabolites (M + 4). For the graphs of percentage change, the mean value of the left-most treatment group was set to 0%. Immunoblotting analysis probed for total or phosphorylated proteins. For a–f, n = 3 biological replicates (circles). Data are mean ± s.e.m. Statistical analysis was performed using two-tailed Student’s t-tests (c), one-way ANOVA with Tukey test (a and d–f) and two-way ANOVA with Sidak test (b); asterisks (*) indicate significant differences compared with the treatment groups marked with daggers (†) or between treatments indicated with brackets (P < 0.05).
