Fig 1: H19 is elevated in NEPC organoids and cell lines and controls the expression of stem cell and NE markers.Relative H19 expression in A patient-derived organoids from normal (1–2) biopsy, AdPC (1–3), and NEPC (OWCM-154, 155, 1078, 1262) and B AdPC (LNCaP, C4-2B and VCAP), and NEPC cell lines (LASCPC-01 and NCI-H660). C Percent methylation on IGF2/H19-Imprinting control region (H19-ICR) derived by Methyl meter assay in representative control (see Supplementary Methods), AdPC, CRPC, and NEPC cell lines. The normal imprinting range was defined as 40–60% methylation. D Relative RNA expression of indicated genes after Trp53/Rb1 DKO induced by lentiviral transduction of Cre-recombinase in Trp53flox/flox/Rb1flox/flox mouse prostate organoids (Cre-GFP) vs. control transduced organoids (EV-GFP). E Relative RNA expression of H19 and NE markers in LNCaP overexpressing H19 vs. control (EV). F Relative RNA expression of indicated genes in doxycycline (DOX) inducible H19FL C4-2B cells upon DOX treatment (0, 200, 400 ng/mL; 48 h) (see also Fig. 6F). G Heat map of relative RNA expression of indicated genes in control (Lv-Scr) and H19 knockdown (Lv-shH19) NEPC organoids. H Relative RNA expression of indicated genes after H19 knockdown in NCI-H660 cells (Lv-shH19) vs. control transduced cells (Lv-Scr). I Western blot (WB) of SOX2 and NSE in control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 and LASCPC-01. J Relative RNA expression of representative lineage-specific genes in mouse prostate organoids after Trp53/Rb1 DKO induced by lentiviral transduction of Cre-recombinase (Cre-GFP) into Trp53flox/flox/Rb1flox/flox mouse prostate organoids. H19 knockdown is denoted as Cre-GFP + shH19. EV transduced organoids (EV-GFP) were used as controls. #p-values < 0.05 vs. EV-GFP organoids. *p-Values < 0.05 vs. Cre-GFP organoids. K WB of representative NE genes in the Trp53flox/flox/Rb1flox/flox mouse prostate organoids transduced with EV, Cre, Cre+shH19, or shH19. L Representative images showing H&E staining (scalebar 50 µm) and immunostaining (scalebar 20 µm) for Ki67 and CK8 in the same organoids from (J). Arrows represent areas of intensive staining. M Relative RNA expression of luminal markers after H19 knockdown in NCI-H660 cells (Lv-shH19) vs. control transduced cells (Lv-Scr). Data are mean ± SD (A, B, H, M), or mean ± SEM (C, D, E, F, J); n = 3 (B, C, E, F, H, J, M) or n = 4 (A, D) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.