Fig. 3: DOT1L inhibition leads to loss in AR protein levels by decreasing its protein stability.

AR protein levels in (a) LNCaP cells treated with EPZ followed by quantitation (n = 5). b AR protein in VCaP and C42B cells after EPZ treatment. c shControl or shDOT1L transfected LNCaP and C42B cells. d PDX organoids treated with EPZ. e AR protein levels in EV or DOT1L overexpressing LNCaP cells. f Proliferation assay of LNCaP cells with and without DOT1L expression in charcoal stripped media measured using MTS assays for 4 days. g AR western analysis after 50 μg/ml Cycloheximide treatment in LNCaP cells treated with Vehicle or EPZ for 8 days (left). Representative experiment shown. Quantitation of AR protein levels from 3 independent experiments (right). h PSA and AR western blot analysis in LNCaP treated with Vehicle and EPZ for 8 days. Representative image shown. i Percentage of RFP + GFP + cells counted by Flow cytometry after 8 days of Vehicle or EPZ treatment in LNCaP cells transfected with ARE-GFP reporter construct. j GSEA plot of Nelson_Response_to_Androgen geneset enriched in LNCaP cells treated with 1 μM EPZ for 8 days compared to Vehicle treatment. mRNA expression of 6 AR target genes measured by qRT-PCR in LNCaP cells after 8 days of (k) 1 μM EPZ treatment and (l) transduction with shDOT1L or shControl lentivirus. m Relative enrichment of AR at 4 target genes measured by ChIP followed by qPCR in LNCaP cells treated with Vehicle or 1 μM EPZ for 8 days. n ChIP-seq plots of H3K79me2 at two AR target genes in LNCaP cells treated with Vehicle or 1 μM EPZ. Statistical tests: p value determined by two-tailed t test (a, f–i, k–m) corrected for multiple comparisons in (k–m). n = 3 independent experiments (b, c, f, h–m). FDR < 25% (j). Error bars represent S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.