l-CaD is expressed in PCa cell lines, and CALD1 co-expresses with positive regulators of EMT in PCa patient data sets.

A Western blot depicting l-CaD protein expression in commercial PCa cell lines. B Barplots representing CALD1 mRNA expression data in PCa cell lines acquired from Cancer Cell Line Encyclopedia. C Frequency of CALD1 alterations in publicly available PCa patient data sets containing CALD1 mutation and copy number alteration data [67–78]. D Representative Western blot depicting l-CaD expression and barplot depicting pooled densitometry of Western blot bands from three biological repeats after 72 h of 5% CSS culture and 24 h DHT treatment. E Venn diagram showing the concordance of top 1000 mRNAs co-expressed with CALD1 between PCa patient data sets available on cBioPortal. F List of concordant hits of mRNA co-expression with CALD1. G MSigDB (version 7.5) hallmark gene sets that significantly overlap and ten MSigDB GO:BP gene sets with the highest percentage of overlap with the list of mRNA concordantly co-expressed with CALD1.

Loss of l-CaD impairs metastasis and invasion of PCa cells.

A Barplot representing viability in PC3 cells transfected with siNeg1, siNeg2, siCaD-1, or siCaD-2. The mean and standard deviation (SD) of three experiments are shown (ns = not significant and *p < 0.05 as determined by the Mann–Whitney–Wilcoxon two-sided test). B Violin plot of relative spheroid size in PC3 cells (n = 160) transfected with siNeg1, siNeg2, siCaD-1, or siCaD-2 and grown in 3D basement membrane matrix culture. Boxplot indicates median and whiskers indicate 1.5 times interquartile range pooled from three experiments (ns = not significant, **p < 0.01, and ***p < 0.001 as determined by t-test). C Violin plot of relative spheroid size in DU145 cells (n = 527) transfected with siNeg1, siNeg2, siCaD-1, or siCaD-2 and grown in 3D basement membrane matrix culture. Boxplot indicates median and whiskers indicate 1.5 times interquartile range pooled from three experiments (ns = not significant, **p < 0.01, and ***p < 0.001 as determined by t-test). D Representative fluorescent merge images of zebrafish embryos 4 days after yolk sac microinjection of mCherry PC3 cells transfected with siNeg1 or siCaD-1. The injection site is highlighted in white. Metastases are indicated with arrows. E Pie chart showing the percentage of zebrafish embryos with visible metastases 4 days after injection pooled from three experiments (*p < 0.05 as analyzed by Fisher’s exact test). F Western blot depicting expression levels of l-CaD in mCherry PC3 cells transfected with siNeg1, siNeg2, siCaD-1, or siCaD-2 48 h after transfection. G Representative violin plots of relative change in primary tumor size (size 4 days after injection / size 1 day after injection). The tumor area was measured from fluorescent images of zebrafish. Boxplot indicates median, and whiskers indicate 1.5 times interquartile range (ns = not significant as determined by t-test). H Representative fluorescent images of zebrafish embryos 4 days after pericardial microinjection of CellTracker Green labeled DU145 cells transfected with siNeg1 or siCaD-1. The primary tumor is highlighted in white. Metastases are indicated with arrows. I Pie chart showing the percentage of zebrafish embryos with visible metastases 4 days after injection to the pericardial cavity (**p < 0.01 as analyzed by Fisher’s exact test). J Western blot depicting expression levels of l-CaD in DU145 cells transfected with siNeg1 or siCaD-1 48 h after transfection. K Violin plots of relative change in primary tumor size (size 4 days after injection/size 1 day after injection). The tumor area was measured from fluorescent images of zebrafish. Boxplot indicates median, and whiskers indicate 1.5 times interquartile range (ns = not significant as determined by t-test). L Representative brightfield and fluorescent merge images of zebrafish embryos 1 day after microinjection of CellTracker Green labeled DU145 cells transfected with siNeg1 or siCaD-1 to the common cardinal vein. Metastases are indicated with arrows. M Box and jitter plots depicting the number of metastases in zebrafish embryos (n = 96). Boxplot indicates median, and whiskers indicate 1.5 times interquartile range (****p < 0.0001 as determined by t-test).

l-CaD expression is upregulated by GR activation promoting growth in organotypic cell culture.

A Correlation between GR and CALD1 mRNA in patient-derived organoids (cBioPortal) [79]. B Western blot depicting GR expression in commercial PCa cell lines. C UCSC genome browser visualization of ReMap ChIP-seq tracks for AR and GR in CALD1 with ENCODE candidate promoter-like signatures highlighted in red and enhancer-like signatures highlighted in yellow and orange. D Barplots depicting l-CaD protein expression in PC3 cells treated with Dex (0.1 µM, 1 µM, or 10 µM) after 24 or 48 hours. The mean and SD of three experiments are shown (ns = not significant, *p < 0.05, and ****p < 0.0001 as determined by t-test). E Barplots depicting l-CaD protein expression in DU145 cells treated with Dex (0.1 µM, 1 µM, or 10 µM) after 24 or 48 hours. The mean and SD of three experiments are shown (ns = not significant, *p < 0.05, and ****p < 0.0001 as determined by t-test). F Barplots depicting l-CaD protein expression in DU145 cells treated with prednisolone (0.1 µM, 1 µM, or 10 µM) after 48 hours. The mean and SD of three experiments are shown (ns = not significant and *p < 0.05 as determined by t-test). G Violin plots of relative spheroid size in PC3 cells transfected with siNeg1 or siCaD-1 and grown in the presence or absence of 1 µM Dex in basement membrane matrix. Boxplot indicates median and whiskers indicate 1.5 times interquartile range pooled form three experiments (ns = not significant, *p < 0.05, and **p < 0.01 as determined by t-test). H Representative brightfield image of day 5 organotypic culture of PC3 cells treated with siNeg-1 or siCaD-1 and exposed to 1 µM Dex or vehicle (DMSO) on day 3 of culture in basement membrane matrix.

l-CaD colocalizes with actin in filopodia, and the knockdown of l-CaD downregulates N-cadherin

A Representative fluorescent merge images of PC3 cells transfected with siNeg1 or siCaD-1 and stained with antibodies recognizing actin and l-CaD. The lower row shows separate images of actin, l-CaD, and merge in a close-up of the upper siNeg1 image. B Representative fluorescent images of PC3 spheroids transfected with siNeg1, siNeg2, or siCaD-1, stained with N-cadherin, and counterstained with DAPI. C Barplots depicting N-cadherin protein intensity in spheroids formed by transfected PC3 cells shown in B. Average was calculated from multiple individual spheroids (N = 22) measured for mean intensity. The mean and SD are shown (ns = not significant, *p < 0.05, and **p < 0.01 as determined by t-test). D Representative fluorescent images of DU145 spheroids transfected with siNeg1, siNeg2, siCaD-1, or siCaD-2 stained against N-cadherin. E Barplots depicting N-cadherin protein intensity in spheroids formed by transfected DU145 cells shown in D. Average was calculated from multiple individual spheroids (N = 30) measured for mean intensity. The mean and SD shown (ns = not significant, ***p < 0.001, and ****p < 0.0001 as determined by t-test). F Representative fluorescent images of DU145 spheroids transfected with siNeg1, siNeg2, siCaD-1, or siCaD-2 stained against ZEB1. G Barplots depicting ZEB1 protein intensity in spheroids formed by transfected DU145 cells shown in F. Average was calculated from multiple individual spheroids (N = 58) measured for mean intensity. The mean and SD shown (ns = not significant, **p < 0.001, ***p < 0.001, and ****p < 0.0001 as determined by t-test).

l-CaD expression is upregulated during enzalutamide resistance in vivo.

A Representative Western blot depicting l-CaD expression and barplot depicting pooled densitometry of Western blot bands from three biological repeats after treatment with enzalutamide (5 days, 10 µM) and Dex (24 h, 0.1 µM). B Scatterplots depicting Pearson correlation between CALD1 and GR or KRT8 mRNA from enzalutamide-treated VCaP xenograft mice. C IHC of l-CaD in castration-resistant VCaP xenografts during enzalutamide response (left) and after attained enzalutamide resistance (right). Individual mice are denoted using Roman numerals. D IHC of GR in castration-resistant VCaP xenografts during enzalutamide response (left) and after attained enzalutamide resistance (right). Individual mice are denoted using Roman numerals. E IHC of l-CaD and GR from adjacent slices of VCaP xenograft tumors in mice during apalutamide resistance. The lower row shows a higher magnification image of the area highlighted on the upper row. Individual mice are denoted using Greek letters. F Scatterplots depicting Pearson correlation between CALD1 and GR or KRT8 mRNA from vehicle-, enzalutamide-, and apalutamide-treated VCaP xenograft mice. G Western blot of vehicle- and apalutamide-treated castration-resistant VCaP xenografts. Quantification of the GR and l-CaD signal relative to actin loading control signal and the average vehicle-treated signal is shown below.