resource source identifier dneasy blood tissue kit qiagen 69504 dynabeads tm myone tm streptavidin t1 invitrogen 65601 pierce tm recombinant protein l  (Thermo Fisher)

Journal: bioRxiv

Article Title: SOX2 utilizes FOXA1 as a heteromeric transcriptional partner to drive proliferation in therapy-resistant prostate cancer

doi: 10.1101/2025.07.18.664790

Figure Lengend Snippet: A) All candidate TFs within 20bp of SOX2 peak in CWR-R1 cells. The e-value is the lowest p-value of any spacing of the secondary motif times the number of secondary motifs; it estimates the expected number of random secondary motifs that would have the observed minimum p-value or less. FOXA1 identified in red. The FOXA1-SOX2 motif enriched by ChIP-seq shows a potential direct interaction on the chromatin. B-F) Gene network signature analyses in PCa patient tumors using the Algorithm for Linking Activity Networks (ALAN) model. Datasets from publicly available sources are depicted across contexts of prostate cancer (GTEX, TCGA, SU2C, and Adeno and NEPC). Gene network signature for FOXA1 (orange) is visualized compared to the gene networks of AR (blue) and SOX2 (red) in each dataset where the median ALAN profile score for each gene network is noted with a black line. Overall network analysis is represented as positive for a median ALAN profile score above 0 and negative for a median ALAN profile score below 0. G) Proximity ligation assay (PLA) measures co-localization (red) of SOX2 and FOXA1 CWR-R1 castration-resistant prostate adenocarcinoma cells. H) Reciprocal co-immunoprecipitation of SOX2 and FOXA1 in CWR-R1 cells. I) Co-immunoprecipitation of FOXA1 with SOX2 in the NEPC cell line NCI-H660. J) Split nano-luciferase complementation reporter assay (Nano-BiT) demonstrating a specific interaction between SOX2 and FOXA1 in HEK293T cells. Tagged AR and FOXA1 were shown to interact as a positive control, and co-transfection of tagged AR and SOX2 were a negative control. Data points represent mean luminescence +/- SD. K) Lentiviral SOX2 overexpression (OE) ablates the AR-FOXA1 Nano-BiT interaction in HEK293T cells. Data points represent mean luminescence +/- SD. L) Lentiviral AR overexpression (OE) ablates the SOX2- FOXA1 Nano-BiT interaction in HEK293T cells, specifically in ARSI conditions. Data points represent mean luminescence +/- SD. M) Nano-BiT in prostate adenocarcinoma LNCaP cells demonstrates strong SOX2-FOXA1 interaction. No significant changes were observed across different AR-signaling contexts. Data points represent mean luminescence +/- SD. N) Western blots of CWR-R1 protein lysate from cells that underwent AR-pathway modulation. Protein bands were quantified using Empiria Studio.

Article Snippet: Co-immunoprecipitation was performed on cells seeded at a density of 1.0 x 10 6 cells per IP using a Dynabeads Protein G Immunoprecipitation Kit (ThermoFisher, Cat: 10007D).

Techniques: ChIP-sequencing, Activity Assay, Proximity Ligation Assay, Immunoprecipitation, Luciferase, Reporter Assay, Positive Control, Cotransfection, Negative Control, Over Expression, Western Blot