Figure 5

LXN indirectly induces a pro-inflammatory trancriptome signature. Lentiviral particles containing Mock control or LXN both with a GFP reporter gene were used to track overexpression of proteins after viral transduction. (A) Representative image of GFP expression in live prostate cells 36 hrs after viral transduction using an epifluorescence microscope. (B) Western blot data depicting a time-course to determine the earliest time after lentiviral transduction at which LXN is overexpressed. α-tubulin serves as a loading control. (C) Representative scatter plot to show differentially expressed genes 36 hrs after transduction of LXN (cut off was ± 2-fold change p < 0.05). LXN was found to be overexpressed 118.31-fold, p = 0.0075, (n = 4) at this time-point after viral transduction. (D) Bright-field images depicting primary prostate cells 48 & 72 hrs after selection in puromycin. Cells were transduced with control particles (Mock) or LXN lentiviral particles both containing a puromycin resistance cassette. Non-transduced primary prostate epithelia (UT) demonstrate the success of viral transduction. (E) Lentiviral transduced cells were confirmed on average to be overexpressing LXN using RT-PCR (418 ± 87.92-fold, p = 0.0032, n = 4). (F) Western blot demonstrating that lentiviral transduced primary prostate epithelia overexpress LXN protein compared to cells transduced with control particles (α-tubulin serves as a loading control). (G) Representative scatter plot to show differentially expressed genes 1 week after transduction of LXN (cut off was ± 2-fold change, p < 0.05). (H) Representative heatmap to demonstrate the largest gene expression changes in individual samples. (I) Funtional annotation of differentially expressed genes in response to LXN overexpresion (see Supplementary Table 1 for detailed list). (J,K,L) Independent validation of the effects of LXN overexpression on CYP1A1, LAMP-3 and ALDH3A1 in non-malignant primary prostate epithelia (n = 9) (M). ALDH1A3 mRNA expression was not found to be significantly affected by LXN overexpression. (N) Stable overexpression of LXN in the luminal-like cancer cell line LNCaP resulted in significant induction of CYP1A1 mRNA expression compared to untransduced cells (UT) or cells transduced with control particles (6.42 ± 0.03 -fold, p < 0.000, n = 3). mRNA expression is presented as fold change relative to cells transduced with control particles (Mock).