Fig 2: Localization, RNA, and protein expression analysis of normal and mutated BEST1. (A) Confocal immunofluorescence images of monolayers from control, BD, ARB, and ADVIRC hiPSC-RPEs after five weeks growth on Corning Transwell filter inserts using a-BEST1 and a-ß-catenin antibodies. Scale bar: 20 µm. (B–C) Quantification of BEST1 (B) and RPE65 (C) expression by qRT-PCR of total mRNA extracted from BD, ARB, and ADVIRC hiPSC-RPEs relative to control (+/+ #1). Independent samples (n = 3) were measured in triplicates and normalized to HPRT1. Data were given as mean ± SD. Please note that Supplementary Figure S2 depicts individual variation of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) measurements between individual control cell lines. (D) Representative Western blot images of whole cell lysates from control, BD, ARB, and ADVIRC hiPSC-RPEs using a-BEST1 antibody. Anti-ß-actin was used as a loading control. (E) Quantification of BEST1 protein expression from (D) relative to control (+/+ #1) and normalized against ß-actin from the same blot. Mean values are given as mean ± SD from two technical replicates per individual sample (n = 2–3). A summary of hiPSC clones used in the experiments is given in Supplementary Table S4. Statistical analyses were performed applying Kruskal-Wallis test, following post-hoc Dunn’s multiple comparisons test including Benjamini-Hochberg procedure: * = p < 0.05; ** = p < 0.01; *** = p < 0.001 and indicated relative to control (+/+ #1).

Fig 3: M2 macrophages secrete higher CXCL5 levels upon crosstalk with CAFs which in turn promote PCa progression via EMT.a Immunofluorescence staining of a-SMA and FAP in prostate stroma CAFs. The number of a-SMA and FAP positive cells in the prostate stroma were markedly increased in the dCko mice compared with the control mice (*P < 0.05. two-tailed Student’s t-Test). Scale bar: 50 µm. b Upper panel: schematic diagram of co-culture system. PCa cells were cultured in the upper compartment and the target cells (NFs) in the lower compartment of the well. Lower panel: WB analysis shows NFs are markedly activated when co-cultured with HIC1-deleted C4-2B cells, and these effects could greatly be abolished by using TGF-ß neutralizing antibody. c Schematic diagram of the conditional culture system. CD14+ PBMCs culture with TGF-ß for 5 days followed by 2 days of conditional culture with (1) IgG, (2) CXCL12, (3) CAF1 conditional medium (CM) or (4) CAF1CM with CXCL12 neutralizing antibody. Then transfer these cells into normal medium for 2 days. Discard the cells and collect the supernatant for cytokine arrays at the end of the culture. d, e Supernatant cytokine array results show CXCL5, CCL2 and MMP-9 were significantly increased in the CM of both CXCL12 activated-M2 macrophages and CAF1 activated-M2 macrophages compared with the corresponding control groups. f Treated with rhCXCL5 could promote prostate organoid growth, and this effect was markedly inhibited by SB225002. g–i The migration and invasive capabilities of PC3 cells were evaluated using a Transwell® assay (24 wells; Matrigel gel; Corning, Inc.). Transwell experiments (g) and WB analysis (h, i) show treatment with rhCXCL5 enhanced the migration and invasive capabilities of PC3 sgHIC1 cells, and activation the AKT signaling pathway. These effects were partially abolished by SB225002. Knockdown CXCL5 receptor-CXCR2 in PC3 cells by siRNAs inhibited the EMT process and the AKT pathway (i). j The EMT phenotype of prostate organoids was also observed after treating with different concentrations of rhCXCL5 and markedly inhibited by SB225002. k ELISA analysis show dCKO mice have higher serum CXCL5 level than control group mice (n = 11 each group) (**P < 0.01, Mann–Whitney U-test).

Fig 4: (A, B) Schematic diagram of the method used to capture circulating tumor cells with a Transwell membrane (Corning Inc., Corning, NY, USA).

Fig 5: CD44 knockdown negatively regulates the properties of CSCs and the expression of p-ERK and Oct4A. the expression of CD44 mRNA in MKN45 cells transfected with CD44 shRNA or scramble shRNA (Ctrli) was detected by qRT-PCR. B. spheroid colony formation ability was reduced by CD44knockdown in MKN28 GC cells. After nearly 3 weeks of culture, CD44 knockdown cells produced significantly fewer spheroid colonies than control cells (transfected with scramble shRNA). C. CD44 knockdown cells displayed elevated chemo-sensitivity. The CCK8 assay was used to evaluate the sensitivity of cytotoxic agent 5-Fu between CD44 knockdown cells and control cells. D. CD44 knockdown cells have reduced migration ability. Cell migration ability was analyzed using the Corning Transwell chamber. The cells were photographed (upper panel) and quantified (lower panel). E. CD44 knockdown cells are more chemo-sensitive. The CCK8 assay was used to evaluate the sensitivity to cytotoxic agent irinotecan (IRI) between CD44 knockdown cells and control cells. F. CD44 knockdown downregulates the expression of p-ERK and Oct4. The expression of CD44, Oct4, phosphorylated ERK1/2(pERK), SOX2, Gli1, phosphorylated AKT(pAKT), total AKT(AKT), and actin in control and CD44-knockdown cells was detected by Western blot. (data are represented as mean +/- SD)