Differential expression (DE) genes used in GSEA were ranked by log2(fold change)

Differential expression (DE) genes used in GSEA were ranked by log2(fold change). estrogen deprivation (LTED) cells. Physique S8. Dose response of fatty acid synthesis and -oxidation inhibitors in long-term estrogen deprivation (LTED) cells. Physique S9. The abrogation of sterol regulatory element-binding proteins (SREBPs) in SUM44 long-term estrogen deprivation (LTED) cells. Physique S10. Expression of sterol regulatory element-binding proteins (expression analysis in clinical specimens from a neo-adjuvant endocrine trial showed a significant association between the increase of expression and lack of clinical response, providing further support for a role of SREBP1 in the acquisition of endocrine resistance in breast malignancy. Conclusions Our characterization of a unique series of AI-resistant ILC models identifies the activation of key regulators of fatty acid and cholesterol metabolism, implicating lipid-metabolic processes driving estrogen-independent IITZ-01 growth of ILC cells. Targeting IITZ-01 these changes may show a strategy for prevention and treatment of endocrine resistance for patients with ILC. Electronic supplementary material The online version of this article (10.1186/s13058-018-1041-8) contains supplementary material, which is available to authorized users. siRNA and 1 pmol siRNA or with 2 pmol non-target siRNA. SiRNA sequences are provided in Additional file?2: Table S1. Q-RT-PCR RNA was extracted with a Qiagen RNeasy kit (74,106; Qiagen, Hilden, Germany). iScript reverse transcription supermix (1,708,841; Bio-Rad Laboratories, Hercules, CA, USA) was used IITZ-01 to generate cDNA. Quantitative polymerase chain reaction (PCR) was then carried out with a CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories) using SsoAdvanced SYBR Green Grasp Mix (Bio-Rad Laboratories). was used as the internal control to normalize gene expression. Primer sequences are provided in Additional file?2: Table S1. Immunoblotting For whole cell lysis, cells were lysed with RIPA buffer supplied with Halt Protease and Phosphatase inhibitor (78,842; Thermo Fisher Scientific, Waltham, MA, USA). Nuclear proteins were extracted with NE-PER? Nuclear and Cytoplasmic Extraction Reagents (78,833; Thermo Fisher Scientific) in accordance with the instructions of the manufacturer. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. Protein bands were detected by fluorescence with Odyssey CLX imaging system (LI-COR Biosciences, Lincoln, NE, USA). The following primary antibodies were used: anti-ER (8644; Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), anti-SREBP1 (SC-13551; Santa Cruz; dilution 1:200), anti–actin (A5441; Sigma-Aldrich; dilution 1:2500), and anti-FASN (3180S; Cell Signaling Technology; dilution 1:1000). Anti-PCNA (NA03; EMD Millipore, Billerica, MA, USA; dilution 1:1000) was kindly provided by Yi Huang (UPMC Hillman Cancer Center) and used as the internal control for nuclear protein. RNA-sequencing and differential expression analysis Parental and LTED MM134 and SUM44 cells were seeded in triplicates in six-well plates. Parental cells were hormone-deprived for 3 days before cell collection. RNA was isolated by using an Illustra RNAspin IITZ-01 Mini Kit (25C0500-72; GE Healthcare, Little Chalfont, UK). RNA-sequencing (RNA-Seq) was carried out by Illumina HiSeq 2000. Natural sequence data were mapped to hg38 genome (ensemble release version 82) and gene counts were quantified with Salmon (version 0.6.0) [20] using default settings. RNA-Seq mapping rates are provided in Additional file?3: Table S2. Differentially expressed (DE) analysis was performed with R package DESeq2 [21] in MM134 cells and SUM44 cells independently. DE genes in individual LTED variants were called using the following criteria: absolute log2(fold change) > log2(1.5) and Benjamini-HochbergCadjusted value of less than?0.001. The complete list of DE genes KLF1 is available in Additional file?4: Table S3. RNA-Seq natural sequence data are available via “type”:”entrez-geo”,”attrs”:”text”:”GSE116744″,”term_id”:”116744″GSE116744 from gene expression omnibus (GEO) (http://ncbi.nlm.nih.gov/geo/). The gene expression (microarray) data of SUM44 tamoxifen-resistant (SUM44 TamR) and parental cells (SUM44PE) were downloaded from GEO [“type”:”entrez-geo”,”attrs”:”text”:”GSE12708″,”term_id”:”12708″GSE12708]. Probes with the highest interquartile range were selected for genes that matched to multiple probes. DE analysis was performed with R package Limma [22], and a Benjamini-HochbergCadjusted value IITZ-01 of less than?0.05 was used to call DE genes in SUM44 TamR cells. Heatmap clustering The Salmon output of gene-level transcript per million (TPM) counts was used, first transforming by log2 (TPM + 1). The top 1000 most variable genes in MM134 or SUM44 cells (by interquartile range) were used for the heatmap. Relative expression values were calculated as fold change to the average expression level in parental cells. Hierarchical clustering of genes was conducted by using the heatmap.3 function (https://natural.githubusercontent.com/obigriffith/biostar-tutorials/grasp/Heatmaps/heatmap.3.R) under R version 3.2.2. The relationship between genes in terms of expression patterns across different samples was quantified with a Euclidean distance measure and visualized with complete-linkage clustering. Pathway analysis Pathway analysis was conducted with Ingenuity Pathway Analysis (IPA) using genes that were differentially expressed in at least three MM134 LTED variants or both SUM44 LTED variants. Complete pathway analysis results are shown in Additional file?5: Table S4. function in Gene Set Enrichment Analysis (GSEA) (version 2.2.2, Broad Institute, Cambridge, MA, USA) was performed using the Reactome cholesterol synthesis signature (Additional file?5: Table S4), downloaded from the Molecular Signature Database (MsigDB, version 6.0, Broad Institute). DE genes ranked by their log2(fold change) were used.