Fig 2: SREBP-2 promotes PCa tumorigenicity and metastasis in vivoA. CWR22Rv1 shNT and shSREBP-2#1 cells were subcutaneously injected into male nude mice (5 mice for each group). The tumor volumes of xenograft were measured and monitored at indicated time. *P < 0.05. B. The weights of subcutaneous CWR22Rv1 shNT and shSREBP-2#1 tumors 31 days after injection. **P < 0.01. Tumor images were shown in Supplementary Figure S7B. C. Representative images and the percentage of Ki67 positive cells in CWR22Rv1 shNT and shSREBP-2#1 tumor tissues. Scale bar = 20 µm, ***P < 0.001. D. H&E and IHC analysis of SREBP-2, c-Myc, ALDH1A1 and CD44 expression in CWR22Rv1 shNT and shSREBP-2#1 tumor tissues. Scale bar = 20 µm. E. Quantitative analysis of SREBP-2, c-Myc, ALDH1A1 and CD44 was performed and reported as total IHC score by assessing the intensity of each staining and the percentage of positive cells, shown as the mean ± SD from each group. *P < 0.05, **P < 0.01, ***P < 0.001. F. representative BLI images of mice bearing metastatic CWR22Rv1 shNT or shSREBP-2#1 tumors 5 weeks after intracardiac injection. G. normalized BLI signals of metastatic CWR22Rv1 shNT or shSREBP-2#1 tumor development over the course of 5 weeks. Data represent the mean ± SEM (N = 8). *P < 0.05.

Fig 3: Confocal images of RALDH1 expressing cells in postnatal human ocular tissue after immunolabeling with an anti-RALDH1 antibody. (A) Low power magnification of ocular tissues demonstrating RALDH1 labeling (red) in the retina and choroid. (B) Negative control slide (non-immune IgG used in place of primary antibody) demonstrating no labeling in the retina and choroid and auto-fluorescence in the RPE. (C, D) Choroid sections demonstrating RALDH1 labeling in extravascular cells throughout the choroidal stroma (arrowhead). Upward arrow in (C-D) indicates orientation for the scleral side of the choroid. Nuclei were counterstained with DAPI (blue). BV, blood vessel; C, choroid; RPE, retinal pigment epithelium; ONL, outer nuclear layer; INL, inner nuclear layer; ILM, inner limiting membrane. Scale bars = 100 µm in A, B; 20 µm in C, D.

Fig 4: SREBP-2 induces PCa stem cell population increase and prostasphere formationA. Expression of c-Myc, ALDH1A1, CD44, NANOG and SOX-2 in LN-Vec, LN-S2#1 and LN-S2#2 cells was determined by qPCR. Data were normalized by ß-actin and represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. B. Percentage of ALDHhigh cells was assessed in LN-Vec, LN-S2#1 and LN-S2#2 cells by flow cytometry following the instructions of the ALDEFLUOR kit. Overexpression of SREBP-2 resulted in a significant elevation of the ALDHhigh cell population compared with control cells. Data were shown as the mean ± SD of three independent experiments. **P < 0.01, ***P < 0.001. C. Primary and secondary prostasphere formation of LN-Vec, LN-S2#1 and LN-S2#2 cells. ***P < 0.001. Representative images of prostaspheres were shown on the bottom. Scale bar = 20 µm. D. Percentages of ALDHhigh and CD44+/CD24- cells in CWR22Rv1 shNT and shSREBP-2#1 cells were assessed by flow cytometry using the ALDEFLUOR kit, and anti-CD44 and anti-CD24 antibodies, respectively. Data were shown as the mean ± SD of from three independent experiments, ***P < 0.001. E. the numbers of primary and secondary prostaspheres of CWR22Rv1 shNT, shSREBP-2#1 and shSREBP-2#2 cells. ***P < 0.001.

Fig 5: Western blot analysis and quantification of RALDH1 and RALDH3 in postnatal human ocular tissues. (A) Cytosol fractions from ocular tissues of donors aged 14, 49, and 50 years were immunblotted with anti-RALDH1 or anti-RALDH3. Top panel: RALDH1 (~ 55–57 kDa) was abundantly expressed in the lens and choroid, moderately expressed in the sclera, and faintly detected in the retina and RPE. Bottom Panel: RALDH3 was not detected in postnatal ocular tissues at any of the ages examined. 3 µg total protein/lane was loaded on the blot. 1.25 µg recombinant human RALDH3 (R3) was loaded as a positive control. (B) Abundance of RALDH1 was measured as the integrated optical density (IOD) per 3 µg total protein of RALDH1-immunopositive bands. (C) Average RALDH1 abundance (± s.e.m.) in ocular tissues of all donors presented in (B) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by Tukey-Kramer test for multiple comparisons).