Mendelian transmission of Rosa26-targted CAGs-rtTA3 transgenes.

<p>*Numbers represent: observed (expected) from heterzygote x wild-type crosses.</p

GFP induction and mKate2 expression is uniform in most organs of <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice.

<p>Immunofluorescence stains for GFP and mKate2 in the small intestine and pancreas of ‘no rtTA’, <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> mice following 1 week of doxycycline treatment. All rtTA strains show strong GFP induction in small intestine (<b>A</b>), but only <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show robust and uniform GFP expression (and mKate2 for <i>RIK</i>) in the pancreatic acinar tissue (<b>B</b>).</p

Adenoviral Cre induces mosaic activation of rtTA and GFP induction in <i>CAGs-LSL-rtTA3</i> and <i>CAGs-LSL-RIK</i> animals.

<p><b>A</b>. Immunofluorescent stains for GFP and mKate2 in liver sections of <i>TG-Ren.713;CAGs-LSL-rtTA3</i> and <i>TG-Ren.713;CAGs-LSL-RIK</i> mice 1 week following intravenous injection of Adenoviral Cre (5×10⁸ PFU) or PBS (<i>CAGs-LSL-RIK</i> only – left panel) and dox treatment. Double transgenic mice exposed to AdenoCre show mosaic expression of GFP (<i>CAGs-LSL-rtTA3</i>) or GFP and mKate2 (<i>CAGs-LSL-RIK</i>). No GFP of mKate2 expression was observed in animals not exposed to Cre. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in lung sections of triple transgenic mice (<i>CAGs-LSL-rtTA3 or RIK;TG-Ren.713;LSL-Kras^G12D</i>). Kras^G12D-induced lung adenomas show strong expression of GFP and mKate2. Lowe panel: higher magnification of the lesion. White arrows indicate rare cells that show mKate2, but not GFP expression.</p

CAGs-rtTA3 and CAGs-RIK show strong expression in adult tissues.

<p>Whole mount epifluorescence images of small intestine, skin, pancreas kidney and liver from <i>R26-rtTA</i>, <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> transgenic animals (all containing <i>TG-Ren.713</i>). <i>R26-rtTA</i> shows strong expression in intestine and skin but weak or patchy expression in most other solid organs. <i>CAGs-rtTA3</i> and <i>CAGs-RIK</i> show almost identical expression patterns in adult mice. <i>CAGs-RIK</i> mice show strong and consistent expression of mKate2.</p

<i>CAGs-LSL-RIK</i> enables tissue-restricted expression of <i>TRE</i>-transgenes in transgenic models of disease.

<p><b>A</b>. Whole mount epifluorescence (top panel) and immunofluorescence images from a quadruple transgenic (<i>CAGs-LSL-RIK;TG-Ren.713;LSL-Kras^G12D;Pdx1-Cre</i>) animal, showing induction of GFP and mKate2 in both normal acinar tissue and pre-neoplastic, Kras^G12D-induced PanIN lesions (top arrow). As observed in AdenoCre treated lungs, some PanIN lesions did not show GFP or mKate2 staining suggesting incomplete LSL excision in a small proportion of cells. <b>B</b>. Immunofluorescent stains for GFP and mKate2 in mammary tissue of <i>CAGs-LSL-RIK;TG-Ren.713;MMTV-Neu;WAP-Cre</i> transgenic mice treated with dox.</p

Additional file 1: Table S1. of Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models

The sequence of sgRNA and oligos used to generate T47D ESR1 mutant cell lines via CRISPR. Table S2 DNA sequence of the oligos used to generate MCF7 ESR1 mutant cell lines via AAV. Table S3 Sequence of the primers used for qPCR assay. Table S4 List of all ligand-independent genes differentially regulated in mutant cells vs WT (FC >2, p < 0.005). Table S5 Disease and function pathways enriched in mutant cells in the absence of estrogen. The novel ligand-independent genes, which were differentially regulated in mutants of each cell line, were pooled and submitted for IPA pathway analysis. The top five relevant functions that were statistically significant are presented in this table. (ZIP 266 kb

Additional file 2: Figure S1. of Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models

Sanger sequencing shows the insertion of Y537S (A > C) and D538G (A > G) in T47D and MCF7 cells. Figure S2 Total ER and phospho ER blotting in all clones of T47D and MCF7 cell lines. a Quantification of P-ER(S118) bands from three independent experiments. Band densities were calculated by ImageJ. P-ER values were corrected to total ER level, and then normalized to vehicle-treated WT groups. b T47D and MCF7 WT or mutant individual clones were hormone-deprived and treated -/+ 1 nM of E2 for 24 hand IB was performed for ER and p-ER at Ser118 site. B-actin was used as a loading control. c Post-hormone-deprived MCF7 or T47D clones were treated with 1 nM of E2 combined with or without 1 μM of Ful for 24 h. RT-qPCR was done using PGR primers. One-way Anova was performed between the basal expression of PGR in each mutant clone and the average expression of PGR in the WT clones (*p < 0.05, **p < 0.01, red) and Student’s t test was used to compare the response before and after fulvestrant treatment (*p < 0.05, **p < 0.01, black). Figure S3 Lack of significant AR overexpression in MCF7 and T47D ESR1-mutant cells: log2 TPM expression of AR in MCF7 and T47D cells based on RNA-seq experiment. b The post-hormone-deprived MCF7 or T47D cells (pooled) were treated with 1 nM E2 combined with or without 1 μM of fulvestrant (ICI) for 24 h. RT-qPCR was done using AR-specific primers. b Immunoblots of AR expression (CST #5153) in post-hormone-deprived MCF7 or T47D cells. Experiments were performed three times, and AR expression was quantified; bars present average AR expression in mutant relative to WT cells. One-way Anova was performed comparing AR mean expression in each mutant clone with mean expression in the WT clones (ns). Figure S4 The ligand-independent growth of T47D-Y537S clones depends on charcoal-stripped serum (Gibco #12676 serum was used in this experiment). WT or mutant clones were hormone-deprived for 3 days, pooled, and treated with veh or 1 nM E2 for up to 9 days. Figure S5 Dose–response curves for 2D growth were plotted for Y537S and D538G mutants of T47D (a) and MCF7 (b) cells after hormone deprivation for 3 days. The cells were treated with 20 pM E2 + Ful, AZD9496, 4OHT and raloxifene. The dose–response curves were fitted with a nonlinear regression model in GraphPad Prism. This figure is a representative of one individual experiment that was repeated six times with consistent results. All experiments were performed in six biological replicates. Figure S6 PCA analysis of 1000 top variable genes between WT and mutants. The top 1000 most variable genes were selected based on interquartile range. The PCA analysis was performed and plotted using PCA function in R. Figure S7 Heatmap of variable genes (Anova, p < 0.0005, maximum FC >2) in mutants and WT cells. Gene expression TPM was estimated by Salmon package. Anova was then used to identify genes differentially expressed between the samples. Genes with a p value <0.0005 and FC >2 that were differentially regulated in at least one mutant vs WT-veh were selected for this heatmap. Figure S8 The post-hormone-deprived MCF7 or T47D cells (pooled or individual clones) were treated with 1 nM of E2 -/+ 1 μM of Ful for 24 h. RT-qPCR was done using GREB1 (a) or IGFBP4 (a) primers. All experiments were performed in three biological replicates. One-way Anova was performed between the basal expressional levels in each mutant clone and the average expression of GREB1 and IGFBP4 in the WT clones (*p < 0.05, **p < 0.01, red) and Student’s t test was used to compare the response before and after Ful treatment (*p < 0.05, **p < 0.01, black). Figure S9 Log2 TPM expression of PGR, GREB1 and IGFBP4 levels in MCF7 and T47D cells based on the RNA-seq experiment. Figure S10. The post-hormone-deprived MCF7 or T47D cells (pooled or individual clones) were transfected with scramble siRNA or ESR1 siRNA for 24 h, and then treated -/+ 1 nM of E2 for 24 h. RT-qPCR was done using ESR1, PGR, or IGFBP4 primers. All experiments were performed in three biological replicates (one-way Anova, *p < 0.05; **p < 0.01). Figure S11 Overlap of novel ligand-independent regulated genes of the ESR1 mutations within one cell line (a) and between the cell lines (b) (chi-square test, **p < 0.01). (PDF 2550 kb