Fig 1: Characterization of LY6D+ Prostate Epithelial Subpopulations from HN Mice(A) FACS analysis of Lin- cells from prostates (from HN mice) confirmed LY6D expression in basal (SCA1int), intermediate (SCA1high), and a small portion of luminal (SCA1low/-) cells. n = 3 for each sorted subpopulation. p values: **p < 0.01 and ***p < 0.001. Error bars represent mean ± SEM.(B) Quantification of keratin expression by IF staining of the FACS-sorted subpopulations as in (A). The number of cells counted in each group is shown from four mice. See also Figure S3A.(C) FACS analysis of LY6D+ cells from HN prostates for their staining patterns of select prostate stem/progenitor and luminal surface markers.(D) Quantification of marker profiles based on FACS subpopulations as in (C). The average of frequency of the indicated populations is from three mice.(E) Comparison of the gene expression profiles from LY6D+ and LY6D- subpopulations (based on RNA-seq data) by principal-component analysis (PCA).(F) Heatmap of differentially expressed genes in sorted LY6D+ and LY6D- subsets from the SCA1high or int or low/- populations. Log2 expression values (based on microarray) were normalized to luminal (SCA1low/-) LY6D- subset for each gene (=0).See also Figure S3.
Fig 2: LY6D Is Associated with Advanced Human PCa(A) Representative IF staining results showing pan-Keratin (pK) (epithelial marker) and LY6D staining in human prostate cancer samples. Scale bars: 20 µm.(B) Kaplan-Meier curve of tissue microarray showing association of LY6D positivity (based on protein) with patient outcomes. The red line depicts patients with positive LY6D (LY6Dpos) expression (n = 14), whereas the blue line patients with negative LY6D (LY6Dneg) expression (n = 51).(C) Kaplan-Meier curve of human prostate cancer (PCa) cohorts (Cancer Genome Atlas Research Network, 2015, Taylor et al., 2010) analyzing time to biochemical recurrence from diagnosis. The red line depicts patients with high LY6D expression (mRNA [z score > 1.3]), whereas the blue line patients with low LY6D expression. Patients with missing disease-free survival status in TCGA; n = 6 are excluded.(D) Kaplan-Meier curve of human TCGA PCa cohort (Cancer Genome Atlas Research Network, 2015) analyzing patients’ overall survival.See also Figure S7.
Fig 3: LY6D Marks CR Luminal Cells that May Possess Regeneration Capacity(A) FACS plots comparing LY6D staining of total Lin- and LY6D+ or - SCA1High or int or Low/- prostate cells from hormone-naive (HN) and castrated (CR) males.(B) Quantification of LY6D expression profiles based on FACS subpopulations as in (A) (HN versus castrated). The average of frequency of the indicated populations is from five mice. p value: *p < 0.05. Error bars represent mean ± SEM.(C) Quantification of the absolute numbers of LY6D+ or - subpopulations based on FACS subpopulations as in (A) (HN versus castrated). The average of frequency of the indicated populations is from five mice. p value: **p < 0.01. n.s., non-significant. Error bars represent mean ± SEM.(D) Schematic diagram showing genetic marking by YFP via tamoxifen-induced activation of CreER.(E) Representative FACS plots showing LY6D staining from tamoxifen-induced K8-CreER;R26Y prostates for Lin- or Lin-YFP+ populations. Tamoxifen induction performed on HN or fully regressed males (after castration) (CR).(F) Representative IF staining results showing YFP and LY6D staining, as well as the co-IF patterns in HN, castrated, and regenerated prostates from K8-CreER;R26Y mice after tamoxifen. Nuclei, DAPI. Arrows depict YFP+LY6D+ double-positive cells. Scale bars: 50 µm.(G) Quantification of the percentage of LY6D+YFP+ per total YFP+ cells from tamoxifen-induced K8-CreER;R26Y prostates. The average of frequency of the indicated populations is from three mice. Error bars represent mean ± SEM.(H) Representative subcutaneous outgrowths from FACS-sorted YFP-marked prostate cells separated as LY6D+ and LY6D- subsets. Note engrafted YFP+LY6D+ cells could generate much larger outgrowth composed of both K5+ (red) and K8+ (green) prostate cells. Scale bars: 50 µm. See also Figures S5H and S5I.See also Figure S5.
Fig 4: LY6D+ Prostate Cells Are Enriched for Organoid-Forming Potential(A) Representative phase images of solid, acinar, and translucent organoids from FACS-sorted Lin-SCA1highLY6D+, Lin-SCA1intLY6D+, and Lin-SCA1low/-LY6D+ prostate cells.(B) Co-IF analysis of organoids derived from FACS-sorted Lin-SCA1highLY6D+, Lin-SCA1intLY6D+, and Lin-SCA1low/-LY6D+ prostate cells for Keratins and p63 expression. Arrow depicts K5+K8+p63+ triple-positive cells. See Figure S4A for individual channels. Scale bars: 50 µm.(C) AR expression in K8+ cells in representative multipotent organoids derived from FACS-sorted Lin-SCA1high LY6D+, Lin-SCA1intLY6D+, or Lin-SCA1low/-LY6D+ prostate cells. Arrows depict AR+K8+ double-positive cells. See Figure S4B for individual channels. Scale bars: 50 µm.(D) Quantification of organoids formed from FACS-sorted LY6D+ and LY6D- (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cell subsets (1,000–3,000 cells/well) from HN mice. n = 5 for each sorted subpopulation. p values: *p < 0.05 and ***p < 0.001. Error bars represent mean ± SEM.(E) Schematic diagram showing organoid culture in the presence or absence of androgen (DHT, dihydrotestosterone).(F) Quantification of organoids derived from FACS-sorted LY6D+ and LY6D- (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cell subsets (1,000–3,000 cells/well) from HN mice, in the presence (+) or absence (-) of DHT. n = 5 for each sorted subpopulation. p value: ***p < 0.001. n.s., nonsignificant. Error bars represent mean ± SEM.(G) Quantification of unipotent (K5+p63+) and multipotent (K8+ K5+p63+) organoids formed from FACS-sorted LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cell subsets (1,000–3,000 cells/well) from HN mice, in the presence (+) or absence (-) of DHT. n = 3 for each sorted subpopulation.(H) Representative co-IF analysis of LY6D, p63, and keratin markers for organoids derived from FACS-sorted LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) prostate cells. Arrows depict double-positive cells. See Figure S4C for individual channels. Scale bars: 50 µm.(I) Androgen-response analysis of CR organoid outgrowth from FACS-sorted Lin-SCA1highLY6D+ subpopulation, in the absence of DHT, only or followed by DHT stimulation, measured by nuclear AR and Ki67 staining. See also Figure S4F. Scale bars: 50 µm.(J) Quantification of nuclear AR expression on LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) derived organoids from castrated mice. Organoids were cultured in the absence of DHT for 14 days, and then stimulated for 7 days with 100 nM DHT. n = 3 for each sorted subpopulation. p values: ***p < 0.001 and **p < 0.01. Error bars represent mean ± SEM.(K) Quantification of Ki67 expression on LY6D+ (Lin-SCA1high, Lin-SCA1int, and Lin-SCA1low/-) derived organoids from castrated mice. Organoids were cultured in the absence of DHT for 14 days, and then stimulated for 7 days with 100 nM DHT. n = 3 for each sorted subpopulation. p value: * p < 0.05. n.s., non-significant. Error bars represent mean ± SEM.See also Figures S3 and S4.
Fig 5: LY6D+ Prostate Cells Are Involved in Prostate Cancer Initiation and Progression(A) Experimental scheme for the lineage-tracing experiment in (B) and (C) showing the time points when K8-CreER;PtenL/L males were castrated, injected with tamoxifen (to induce Pten inactivation), regenerated with androgen, and analyzed.(B) Co-IF staining of HN, regressed, and regenerated K8-CreER;PtenL/L prostates showing overlap of pAKT (green) and LY6D (red) in HG-PIN lesions. Nuclei, DAPI (blue). Scale bars: 50 µm.(C) Co-IF staining showing abundant pAKT+ LY6D+ prostate cancer cells in CR HG-PIN lesions observed in K8-CreER;PtenL/L mice at the second round of regression, with Pten inactivation induced upon surgical castration (i.e., first round regression). VP, ventral prostate; AP, anterior prostate. Scale bars: 50 µm.See also Figure S6.
Supplier Page from MilliporeSigma for Anti-LY6D antibody produced in rabbit