Fig. 3: The proliferation of breast cancer cells depends on the tyrosine phosphorylation of lipin-1.

a, b Re-expression of WT-lipin-1 but not 3YF-lipin-1 restored the proliferation of breast cancer cells. LPIN1-KO MDA-MB-231 cells (a) or MDA-MB-468 cells knocked down of LPIN1(LPIN1-KD) (b) infected with empty vector (ctrl), WT-lipin-1 or 3YF-lipin-1 and were maintained in complete medium containing 10% FBS. The CCK-8 assay (n = 4 experiments) and BrdU incorporation assay (n = 5 experiments) were performed to determine viable cell number. (Left graph of a) ctrl versus WT-lipin-1, *P = 0.0168 (day 3), ***P < 0.001 (day 4); WT-lipin-1 versus 3YF-lipin-1, ^###P < 0.001 (day 4). (Left graph of b) ctrl versus WT-lipin-1, **P = 0.0011 (day 4); WT-lipin-1 versus 3YF-lipin-1, ^###P < 0.001 (day 4). c The soft agar colony formation assay was performed with LPIN1-KO MDA-MB-231 cells or LPIN1-KD MDA-MB-468 cells reconstituted with WT-lipin-1, 3YF-lipin-1 or empty vector as a control. The cells were maintained in complete medium containing 10% FBS. Scale bars, 5 mm. d Xenograft tumour growth in mice. Volumes of tumour burdened in nude mice receiving LPIN1-KO MDA-MB-231 cells reconstituted with WT-lipin-1, 3YF-lipin-1 or empty vector as a control were measured on different days after implantation. n = 6 mice per group. Ctrl versus WT-lipin-1, *P = 0.0461 (day 19), *P = 0.0389 (day 22), *P = 0.0102 (day 25); WT-lipin-1 versus 3YF-lipin-1, ^#P = 0.0161 (day 25). e Representative images and tumour weights of mouse xenograft tumours from d. Scale bar, 10 mm. n = 6 mice per group. f, g Relative levels of TAG (f) and phospholipid (g) of xenograft tumours from nude mice implanted with LPIN1-KO MDA-MB-231 cells reconstituted with WT-lipin-1 or 3YF-lipin-1. n = 9 mice per group. The whole list of lipids identified by lipidomics can be found in Supplementary Data 1. h Schematic diagram of the synthesis of PE and TAG from glycerol-3-phosphate (G-3-P) in mammalian cells. i Knockdown of EPT-1 or inhibition of TAG synthesis impeded the proliferation of breast cancer cells. MDA-MB-231 cells were knocked down of ethanolamine phosphotransferase-1 (EPT-1 KD) and/or treated with the DGAT inhibitors PF-04620110 (DGAT1 inhibitor) and PF-06424439 (DGAT2 inhibitor). The EPT-1 KD MDA-MB-231 cells were infected with HA-EPT-1, and were maintained in complete medium containing 10% FBS. The CCK-8 assays were performed to determine viable cell numbers. Ctrl shRNA versus DGATi + EPT-1 shRNA, *P = 0.0236 (day 3), ***P < 0.001 (day 4); ctrl shRNA versus EPT-1 shRNA, ^##P = 0.0027 (day 4); EPT-1 shRNA versus EPT-1 shRNA + HA-EPT-1, ^†P = 0.0208 (day 4). a, b, i Quantified in each independent experiment. d–g Quantified for each xenograft tumour. Data are mean ± s.e.m.; ordinary two-way ANOVA followed by Tukey in (left graph of a and b, i); ordinary one-way ANOVA followed by Tukey in (right graphs of a and b, e); two-way ANOVA (repeated measure) with Geisser–Greenhouse’s correction, followed by Tukey in d; two-tailed unpaired Student’s t-test in f, g. ***P < 0.001, N.S. not significant. Source data are provided as a Source Data file.