Extended Data Fig. 5. MAF-E2 induced transcriptional changes in BCa cell lines and ER dependency.
a, Principal component analysis (PCA) on the rlog normalized expression matrix (showing first two components). Batch effect was adjusted gene-wise using a linear model. b, RNA-seq expression of a selected gene from each cluster represented by fold-change of RPKM values. Data represents the average of 3 biological replicates. c, Analysis of MAF expression in control and MAF-overexpressing MCF7 and T47D cell lines under hormone deprivation (HD) conditions and after 10 nM E2 administration by qRT-PCR. Expression was normalized to the housekeeping gene GAPDH. n = 3 independent biological replicates. Data are mean ± s.d. d, qRT-PCR expression analysis of a selected gene in each cluster in control (mock-infected) and MAF-overexpressing T47D cells under HD conditions and after 10 nM E2 administration. Expression was normalized to the housekeeping gene GAPDH. MUCL, n = 3; SPANXA1, n = 3; GREB1, n = 5; BMF, n = 4; NFATC4, n = 4; FGF18, n = 7; PTHLH, n = 7; JAG1, n = 7; TMEM2, n = 3; TGFA, n = 3 and JAK1, n = 3. 3 to 7 biological replicates per gene. Data are mean ± s.d. The P value was calculated using a two-sided t-test. e, qRT-PCR expression analysis of selected genes in control (mock-infected) and MAF-overexpressing T47D cells transduced with Androgen Receptor (AR)-targeted shRNA and cultured under HD conditions and after 10 nM E2 administration. Expression was normalized to the housekeeping gene GAPDH. n = 3 independent biological replicates. Data are mean ± s.d. The P value was calculated using a two-sided t-test. f, qRT-PCR expression analysis of selected genes in control (Ad-ctrl) and MAF-overexpressing (Ad-cre) mouse BCa cells transduced with AR-targeted shRNA and cultured under HD conditions or E2 treated (10 nM). Expression was normalized to the housekeeping gene GAPDH. n = 3 independent biological replicates. Data are mean ± s.d. The P value was calculated using a two-sided t-test. g, Immunoblot showing ER degradation by using ER-PROTAC in control (mock) and MAF-overexpressing MCF7 cells 1 and 6 hours after E2 stimulation. GAPDH was used as a loading control. h, Principal component analysis (PCA) on the rlog normalized expression matrix (showing first two components). Batch effect was adjusted gene-wise using a linear model. i, RNA-seq heatmap of differentially expressed genes, already defined clusters 5 and 6, in MAF-overexpressing compared to control (mock-infected) MCF7 cells upon ER degradation followed by administration of E2 (10 nM for 6 h). Color scale indicates expression levels. Red indicates upregulation and blue shows downregulation. n = 3 biological replicates. j, qRT-PCR expression analysis of PTHLH and JAG1 in MAF-overexpressing MCF7 cells transduced with PTHLH and JAG1-targeted shRNAs. Expression was normalized to the housekeeping gene GAPDH. k, Schematic representation of the experimental design. Normalized in vivo photon flux quantification (mean ± sem) of bone metastasis in BALB/c nude mice injected intracardially with control (scramble, n = 14 limbs) and knockdown of PTHLH or JAG1 (n = 14 limbs) in MAF-overexpressing MCF7 cells (left). Normalized ex vivo photon flux quantification of bone metastasis. The median (centre line), first and third quartiles (box limits) and the minimum to maximum values (whiskers) are shown. Statistical significance, two-tailed Mann–Whitney test (right).