Fig 2: LEDGF/p75 and its IBD-interacting partners are differentially upregulated in DTX-resistant PCa cell lines. Upper panels: immunoblots showing upregulation of (A) LEDGF/p75, (B) JPO2, (C) c-MYC, (D) menin, (E) MLL, (F) IWS1, (G) ASK1, (H) PogZ, (I) Med-1, and (J) HRP2 in DTX-resistant PC3-DR and DU145-DR cells compared to their parental, drug-sensitive counterparts. Bottom panels: bar graphs showing quantification of fold change in protein expression from at least three independent experiments per cell line via densitometric ImageJ analysis, with values normalized to β-actin. Paired t-test statistical analysis revealed that protein expression in DTX-resistant PCa cells achieved statistical significance compared to controls (* p < 0.05, ** p < 0.01). Due to the very low expression of some of the proteins in the parental PC3 and DU145 cells, we normalized their expression to an arbitrary value of 0.10 for quantification purposes. Error bars represent mean ± standard deviation (SD).

Fig 3: LEDGF/p75 depletion does not alter the protein expression of selected IBD interacting partners in DTX-resistant cells but affects their nuclear localization patterns. LEDGF/p75 knockdown was assessed by immunoblotting using a rabbit anti-LEDGF/p75 antibody in PC3-DR (A) and DU145-DR (B) cells transfected with siLEDGF/p75 (KD) or scrambled duplex (SD) control oligos. Representative blots show that the protein expression levels of JPO2, c-MYC, menin, MLL, and HRP2 were not altered by LEDGF/p75 depletion. For confocal microscopy analysis, human anti-LEDGF/p75 autoantibodies were co-incubated with rabbit antibodies to JPO2 (C), c-MYC (D), menin (E), MLL (F), and HRP2 (G) in DTX-resistant PC3 cells with and without LEDGF/p75 depletion. Detection of nuclear proteins was achieved with FITC-labeled secondary anti-human antibody (green) and rhodamine-labeled secondary anti-rabbit antibody (red). Merged images show the yellow-orange staining typical of co-localization. DAPI was used for nuclear staining.

Fig 4: Knockdown of LEDGF/p75, JPO2, menin, or HRP2 inhibits the clonogenicity of PC3-DR cells. Representative images of clonogenic assay plates show decrease in colony formation in PC3-DR cells with LEDGF/p75 (A), JPO2 (C), menin (E), or HRP2 (G) knockdowns (KD) compared to scrambled duplex control cells (SD) in the presence and absence of DTX. Colonies were counted after 10 days of treatment. Adjacent bar graphs show quantification of PC3-DR colonies (B,D,F,H) and represent the average of colonies counted in at least three independent experiments. SEM was calculated. Statistical significance was determined by comparing the values for cells transfected with si-SD control oligos with values for cells with LEDGF/p75, JPO2, menin, or HRP2 knockdown in the presence or absence of DTX, using Student’s t-test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Fig 5: Knockdown of LEDGF/p75, JPO2, menin, or HRP2 impairs tumorsphere formation in PC3-DR and DU145-DR cells. siRNA-mediated knockdowns of LEDGF/p75 (A), JPO2 (B), menin (C), and HRP2 (D) were confirmed by immunoblotting in PC3-DR and DU145-DR cells transfected with specific siRNAs (KD) compared to cells transfected with SD control oligos. Representative images of tumorsphere formation assays showing a decrease in spheroid size in PC3-DR and DU145-DR cells after individual siRNA-mediated depletion of LEDGF/p75, JPO2, menin, or HRP2 compared to SD control spheres. Tumorspheres were visualized using phase contrast or Hoffman Modulation Contrast microscopy. Tumorsphere area was quantified (bottom graphs) from triplicate images per experiment using ImageJ software. Data are from at least 3 independent experiments and are represented as mean SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001.