Fig 2: Validation of the specificity of human anti-LEDGF/p75 autoantibodies used in this study. (A) Representative human monospecific (only fluorescence pattern detected) anti-LEDGF/p75 sera (EC10 and PL48) displaying the characteristic dense fine speckled (DFS) nuclear pattern of high titer anti-LEDGF/p75 autoantibodies, also known as anti-DFS or anti-DFS70 autoantibodies (FITC), in HEp-2 antinuclear antibody (ANA) test slides. Pre-adsorption of the two human anti-LEDGF/p75 sera with the autoepitope-containing IBD peptide (LEDGF/p75 residues 347–429) abolished their immunoreactivity in HEp-2 ANA test slides. (B) Immunoblots showing immunoreactivity of the selected anti-LEDGF/p75 sera against a 75 kD protein band in whole cell lysates of both parental and DTX-resistant PC3 and DU145 cell lines, with noticeable increased expression of the protein in the resistant cells. (C) Immunoblots showing the reactivity of representative human serum EC10 against LEDGF/p75 in whole lysates from PC3-DR and DU145-DR cells, with siRNA-mediated LEDGF/p75 knockdown (KD) compared to scrambled duplex control (SD). Note the loss of serum immunoreactivity against the 75 kD protein band in cells with LEDGFp75 knockdown. Beta-actin was used as loading control in panels B and C. (D) Immunoprecipitation of endogenous LEDGF/p75 from PC3-DR and DU145-DR cells using representative anti-LEDGF/p75 human serum EC10. A well-characterized LEDGF/p75 interacting partner, JPO2, was used as a positive control to initially monitor and validate the capacity of monospecific anti-LEDGF/p75 sera to immunoprecipitate its IBD-interacting proteins. Irrelevant normal human serum (NHS) was used as negative control. (E) Pre-adsorption of anti-LEDGFp75 human EC10 serum with the autoepitope-containing IBD polypeptide abolished its ability to co-immunoprecipitate LEDGF/p75 and JPO2, again confirming the specificity of the serum autoantibodies.

Fig 3: AXL expression and GAS6 stimulation in breast cancer cell lines. (A). Scatter plot showing AXL expression levels based on RNA-Seq data and its protein expression based on proteomics data (both from CCLA) as indicated. The color of the dots represents the subtype of breast cancer cell lines: TNBC (red), luminal A (green), luminal B (blue) and HER2+ (yellow). (B). Western blot results showing AXL expression level in a panel of nine TNBC cell lines. Beta-actin serves as a loading control. (C). MDA-MB-231 cells were treated with the indicated concentration (ng/mL) of GAS6 for 10 min. Phosphorylation levels of AXL and AKT and their total protein expression levels were examined by Western blot analysis. (D). MDA-MB-231 cells were treated with 400 ng/mL GAS6 for different time points as indicated. Phosphorylation levels of AXL and AKT and their total protein expression levels were examined by Western blot analysis as shown. (The original western blot images were included in Figure S1).

Fig 4: MYB repression by high dose androgen occurs late due to the reduced stability of its mRNA.A and B, LNCaP and C4-2 cells were incubated with DHT (1 nM and 100 nM) for 6 h prior to the treatment with Actinomycin D. After 1 h, total RNA was isolated at different time intervals for up to 120 min, and MYB mRNA levels were analyzed by qRT PCR. 18s rRNA was used as an internal control. Percent mRNA decay was calculated from the earliest time point of analysis (0 min), and the data was presented as a line graph (mean ± S.D.). C–F, prostate cancer cells were treated with low (1 nM) or high (100 nM) doses of DHT for increasing time intervals (0–48 h). MYB expression was analyzed at both mRNA and protein levels by qRT-PCR and immunoblot assays, respectively. ACTB (qRT-PCR) and β-actin (immunoblot) were used as internal controls. Relative differences in MYB mRNA and protein levels are presented in the bar graph (mean ± S.D.), ∗p = < 0.05. G and H, LNCaP and C4-2 cells were incubated with DHT (1 nM and 100 nM) for 18 h prior to treatment with Actinomycin D. After 1 h, total RNA was isolated at different time points (0–120 min), and MYB mRNA expression was analyzed by qRT PCR. 18s rRNA was used as an internal control. Percent mRNA decay was calculated, and the data was presented as a line graph (mean ± S.D.). ∗p < 0.05; n.s. not significant. DHT, dihydrotestosterone.

Fig 5: Androgen regulates MYB expression in a dose-dependent biphasic manner. Low (LNCaP-Neo/C4-2-shMYB) and high (LNCaP-MYB/C4-2-Scr) MYB-expressing prostate cancer cells were cultured in media supplemented with FBS, and AR expression was examined at transcripts (A) and protein (B) levels using qRT-PCR and immunoblotting, respectively. ACTB (for qRT-PCR) and β-actin (for immunoblot assay) were used as internal controls. Protein band intensity was quantified by densitometry, and the data were plotted as a bar graph. LNCaP and C4-2 cells were grown in a regular medium (supplemented with FBS) to subconfluence. Subsequently, the medium was replaced with fresh FBS- or charcoal-stripped serum (CSS)–containing medium. After 48 h of incubation, MYB expression was analyzed at both mRNA (C) and protein (D) levels. For a dose-response, cells were treated with increasing concentrations of DHT (0–100 nM) for 48 h in CSS-containing media, and MYB expression was examined at mRNA (E) and protein (F) levels. G and H, cells were treated with DHT (0–100 nM) in CSS media and the change in growth measured at different time intervals (48–96 h). The data is presented as line graphs. ∗ indicates statistical significance (p < 0.05), n.s. not significant. AR, androgen receptor; DHT, dihydrotestosterone.