Extended Data Fig. 5. PCK2 phosphorylates ACSL4 at T679.
a, Immunoblots were performed with parental HONE1 cells (sg GFP), ACSL4-knockout HONE1 cells (sg ACSL4), ACSL4-knockout HONE1 cells stably expressing ACSL4 T679A mutation (T679A), treating cell lysates with λ protein phosphatase (λ-phosphatase) or in the presence of blocking peptide for ACSL4 Thr679 phosphorylation (peptide). The p-ACSL4 (T679) antibody was used to detect the phosphorylation level of ACSL4 T679 site. b, IHC analyses of human NPC samples were performed with the p-ACSL4(T679) antibody in the presence or absence of a blocking peptide for p-ACSL4(T679). c-g, LC–MS/MS detection for ACSL4-interacting proteins in ACSL4-containing protein complex immunoprecipitated from HONE1 TRCs and bulk cells. Volcano plot showing the fold change and −log10 (P-value of two-sided Student’s t-test) of differential interaction proteins in HONE1 TRCs and bulk cells. Dotted line indicates factors with fold change > 2 and significance P < 0.05 (c). The peak area and PSMs of PKM2 (d), PFKP (e), PPM1G (f) and PCK2 (g) are shown. n = 4. h, The relative mRNA expressions of indicated genes were analyzed by qRT-PCR for gene knocked-down efficiency in HONE1 cells. P = 0.00005, 0.000040, 0.0000050, 0.000005, 0.00003, 0.000048, 0.000013 and 0.000012. i, Immunoblots showing the expression of ACSL4 and ACSL4 pT679 in HONE1 cells transfected with indicated siRNAs. j, The relative mRNA expression of PCK2 and PCK1 in HONE1 and HK1 cells. P = 0.0001 and 0.0000366. k, PCK2-knockout HONE1 cells and parental cells were treated with or without 3-MPA. Immunoblots showing the expression of p-ACSL4(T679) and ACSL4 in cells. l, Immunoblots showing the expression of ACSL4 and ACSL4 pT679 in HONE1 cells or A375 cells treated with phosphoenolpyruvate (PEP). m, Representative immunofluorescence staining image showing the co-localization of PCK2 (red) and mitochondria (green) in HONE1 cells and HK1 cells stably transfected with mito-GFP. Scale bar: 10 μm. n, Immunoelectron microscopy of ACSL4 localization in mitochondria in HONE1 cells by using a gold-labeled anti-ACSL4 antibody (10 nm gold). Bars: 0.2 μm. o, Subfractionation of highly purified mitochondria from HONE1 cells showing the presence of ACSL4 protein in mitochondria of HONE1 cells and HK1 cells. WCL, whole cell lysate; Cyto, cytosol; OM, mitochondria outer membrane; IM, inner membrane; Mx, mitochondria matrix; IMS, mitochondria intermembrane space. α-Tubulin, Tom20, TIMM22, MnSOD and Cyto C are markers for cytosol, mitochondria outer membrane (OM), inner membrane (IM), matrix (Mx) and inter-membrane space (IMS), respectively. p, Cells expressing ACSL4-mCherry and PCK2-GFP were seeded in 3D fibrin gels (TRCs) or not (bulk cells). Immunofluorescence analysis to detect ACSL4 (red) or PCK2 (green). Scale bar: 10 μm. Data are shown as mean ± SD (d-h, j), unpaired two-tailed t-test (d-g, j) or one-way ANOVA (h), n = 3 independent experiments. One of three experiments is shown (a, i, k-l, o).