Supplementary Materials Physique S1 LIPG mRNA expression in breast cancer cell lines. intracellular and extracellular cell fraction of full media control (FM), unfavorable control (only transfection reagent, NC), empty vector (EV) and LIPG overexpressing (OE) cells. PS: Precision Plus Protein? Dual Color Standard; MM: MagicMark? XP Western Protein Standard. (c) Immunofluorescence of transfected cells with an anti\FLAG antibody targeting the LIPG\FLAG fusion protein encoded by the LIPG overexpression construct (mRNA levels were analysed by qPCR. Lipid droplets were visualized with BODIPY 493/503 staining (green). Nuclei were stained with DAPI (blue). Bar diagrams represent the mean SEM (n?= 3). ***P? 0.001. P\value was calculated by unpaired two\tailed Student’s t\test. Physique S3. mRNA upregulation in senescent MCF\7/NeuT cells results in secretion of LIPG protein. (a) qPCR analysis showing a 15\fold increase in levels Esaxerenone of mRNA in MCF\7/NeuT cells incubated with dox. (b) Representative Western blot showing levels of mature 68?kDa LIPG and its 40?kDa cleaved N\terminal fragment in the supernatant of MCF\7/NeuT cells treated with/without dox and densitometric quantification of Western blot signals of three Esaxerenone independent experiments. (c) Representative Western blot of LIPG in the corresponding cellular lysates showing the remaining cytoplasmic pool of LIPG. For the strongest three signals (57?kDa, 48?kDa and 42?kDa), which could correspond to the non\mature unglycosylated LIPG (57?kDa) and other uncharacterized splice variants, densitometric quantification of Western blot signals is shown for three independent experiments. (d) Immunofluorescence of fixed MCF\7/NeuT cells, treated with/without Triton X\100 for permeabilisation, showing no increase in cytoplasmic LIPG immunoreactivity (mRNA upregulation is not driven by HER2 overexpression (a) Western blots showing phosphorylation of AKT, ERK1/2 and P38 in parental MCF\7 cells and in MCF\7 cells stably transfected with wildtype Her2 and the mutant insYVMAHer2. (b) mRNA expression level in the three cell lines determined by qPCR. expression in parental MCF\7 cells was taken as a reference. As an endogenous control UBC (ubiquitin C protein) was used. The bars represent the mean??SEM (n?= 6). Physique S5. Pharmacological inhibition and silencing of ACC lead to upregulation of expression. (a) qPCR analysis of LIPG mRNA expression in MCF\7 cells incubated for 24?h with TOFA or cerulenin at the indicated concentrations. The bar diagrams represent the mean SEM of two impartial experiments. (b) left: qPCR analysis showing ACACA mRNA levels in MCF\7 cells after transfection with scrambled si\RNA as a negative control (si\neg) and two different si\RNA oligos targeting ACACA (si\ACC\A and si\ACC\B), compared to FM (full media, non\transfected control) and Lipo (Lipofectamine only, mock\transfected). Right: Representative Western blot showing ACC protein levels as well as Calnexin as a loading control, and densitometric quantification Esaxerenone of the ratio (ACC/Calnexin) from Western blot signals of three impartial experiments. (c) qPCR analysis showing LIPG mRNA levels in the same samples as in (b). Bar diagrams represent the mean SEM of three impartial experiments; **P? 0.01; ***P? 0.001. ****P? 0.0001. P\values were calculated by unpaired two\tailed Student’s t\test comparing each of the siRNAs with the unfavorable control. Physique S6. Lipid droplets confer survival advantage under starvation. (a) Cell number after starvation for the indicated time period. In the feeding phase cells were incubated with OA to allow formation of TAG stores, or with solvent only. In the starvation phase cells were transferred to glucose\free and serum\free medium and cell TGFB3 number was monitored for 10 days. (b) Mitochondrial integrity in cells under starvation that Esaxerenone have been previously fed with/without OA, determined by quantification of TMRE fluorescence, normalized to cell number. The bar diagrams represent the mean SEM of three impartial experiments. ***P? 0.001, unpaired two\tailed Student’s t\test. Physique S7: Silencing of LIPG in MDA\MB\468 and MCF\7 breast cancer cells. (a) qPCR analysis showing LIPG mRNA levels in MDA\MB\468 cells after transfection with.