Journal: bioRxiv

Article Title: Stage-Specific Regulation of DNA Damage Repair by the Circadian Regulator, CRY1, in Prostate Cancer

doi: 10.64898/2026.05.19.726303

Figure Lengend Snippet: A. Frequency of CRY1 gene alterations (i.e., amplifications and mutations) in primary and metastatic PCa datasets from cBioPortal—Fred Hutchinson CRPC (Nat Med 2016), TCGA(Cell 2015), MCTP(Nature 2012), and SU2C/PCF Dream Team (PNAS 2019) datasets. B. cBioPortal analyses for overall survival in PCa patients with CRY1 altered versus unaltered groups. C. Cell Titer-Glo 2.0 Cell Viability Assay in LNCaP shCRY1 and upon treatment with CRY1 inhibitor (M47). n=3, ***p<0.001 and ****p<0.0001. D. Cell Titer-Glo 2.0 Cell Viability Assay in C4-2 shCRY1 and upon treatment with CRY1 inhibitor (M47). n=3, **p<0.01 and ****p<0.0001. E. Schematic diagram of experimental design for C4-2 shCRY1 xenograft model. F. Tumor doubling time is significantly increased in C4-2-shCRY1 xenograft model Confirming that CRY1 promotes tumor growth in xenograft model, p<0.05. G. Patient-Derived Explants (PDE) prostate cancer tumor tissue treated with M47 (0, 100, and 150 uM) for 72 h and analyzed for H-I. H&E & Ki67 staining within PDE tissues. Scale bars are 50 & 200 µM.

Article Snippet: The LNCaP and C4-2 PCa cell lines were purchased from the American Type Culture Collection (ATCC), and they provided initial authentication.

Techniques: Viability Assay, Derivative Assay, Staining

Journal: bioRxiv

Article Title: Stage-Specific Regulation of DNA Damage Repair by the Circadian Regulator, CRY1, in Prostate Cancer

doi: 10.64898/2026.05.19.726303

Figure Lengend Snippet: A-B. Workflow showing RNA-seq analysis after CRY1 knockdown in LNCaP cells, MA plot displaying differential gene expression, and Western blot confirming CRY1 knockdown. (A) Pathway enrichment analysis differentially expressed genes in LNCaP cells, KEGG and Hallmark. (B) C. Venn diagram comparing differentially expressed genes between LNCaP and C4-2 cells after CRY1 knockdown, and KEGG pathway enrichment for unique and shared gene sets using Enrichr. p<0.01 D-E. Three Gene Set Enrichment Analysis (GSEA) KEGG pathways and leading-edge plots highlighting specific DDR-related pathways (e.g., base excision repair, mismatch repair, and homologous recombination) that are significantly enriched after CRY1 knockdown.

Article Snippet: The LNCaP and C4-2 PCa cell lines were purchased from the American Type Culture Collection (ATCC), and they provided initial authentication.

Techniques: RNA Sequencing, Knockdown, Gene Expression, Western Blot, Homologous Recombination

Journal: bioRxiv

Article Title: Stage-Specific Regulation of DNA Damage Repair by the Circadian Regulator, CRY1, in Prostate Cancer

doi: 10.64898/2026.05.19.726303

Figure Lengend Snippet: A. Library overview and schematic of DDR CRISPR screen workflow. B. Venn diagram showing overlap of negatively enriched hits (essential DDR genes) between CONtrol and CRY1 KD in LNCaP cells. C. Venn diagram showing overlap of negatively enriched hits (essential DDR genes) between CONtrol and CRY1 KD in C4-2 cells. D. Gene-level RRA enrichment plots highlighting top negatively selected DDR genes under CRY1 KD in LNCaP cells. E. Gene-level RRA enrichment plots highlighting top negatively selected DDR genes under CRY1 KD in C4-2 cells. F-G. Guide RNA plots for targets of interest in LNCaP (PARP1, APLF, MCM5) and in C4-2 (PARP1, ERCC3, ATM). H. Heatmap depicting Log2FoldChange for 20 commonly shared dependencies identified in LNCaP and C42, illustrating increased reliance on specific repair pathways following CRY1 KD.

Article Snippet: The LNCaP and C4-2 PCa cell lines were purchased from the American Type Culture Collection (ATCC), and they provided initial authentication.

Techniques: CRISPR, Control

Journal: bioRxiv

Article Title: Stage-Specific Regulation of DNA Damage Repair by the Circadian Regulator, CRY1, in Prostate Cancer

doi: 10.64898/2026.05.19.726303

Figure Lengend Snippet: A. Venn diagram illustrating overlap of significantly depleted genes (negative RRA selections, p <.05) between LNCaP and C4-2, with associated KEGG pathway enrichment. B. Schematic illustrating unique and shared pathways in HTS (LNCaP) and CRPC (C4-2) prostate cancer models, highlighting candidate DDR and metabolic vulnerabilities. The diagram depicts pathway nodes selectively altered in HTS (APLF and MCM5) and CRPC (ATM and ERCC3), along with available small-molecule inhibitors used or proposed for further functional validation. Kaplan–Meier analyses from cBioPortal are incorporated to compare overall survival in PCa patients with altered versus unaltered expression of APLF and MCM5 (HTS-specific targets) and ATM and ERCC3 (CRPC-specific targets), demonstrating the clinical relevance of these pathway perturbations.

Article Snippet: The LNCaP and C4-2 PCa cell lines were purchased from the American Type Culture Collection (ATCC), and they provided initial authentication.

Techniques: Functional Assay, Biomarker Discovery, Expressing

Journal: bioRxiv

Article Title: Stage-Specific Regulation of DNA Damage Repair by the Circadian Regulator, CRY1, in Prostate Cancer

doi: 10.64898/2026.05.19.726303

Figure Lengend Snippet: A. Relative viability of LNCaP cell with CRY1 knockdown following treatment with PARP inhibitors, a class of DDR inhibitors common to both HTS and CRPC models, assessed by CellTiter-Glo assay. B. Relative viability of LNCaP cell treated with the CRY1 inhibitor M47 in combination with PARP inhibitors, measured by CellTiter-Glo assay. C. Relative viability of C4-2 cell with CRY1 knockdown following treatment with PARP inhibitors, a class of DDR inhibitors common to both HTS and CRPC models, assessed by CellTiter-Glo assay. D. Relative viability of C4-2 cell treated with the CRY1 inhibitor M47 in combination with PARP inhibitors, measured by CellTiter-Glo assay. E. Relative viability of LNCaP cells with CRY1 knockdown treated with HTS-specific DDR inhibitors targeting APLF and MCM5. F. Relative viability of LNCaP cells treated with M47 in combination with APLF and MCM5 inhibitors. G. Relative viability of C4-2 cells with CRY1 knockdown treated with CRPC-specific DDR inhibitors targeting ATM and ERCC3. H. Relative viability of C4-2 cells treated with M47 in combination with ATM and ERCC3 inhibitors. All viability measurements were obtained using the CellTiter-Glo luminescence assay. Bars show mean±SEM of three biological replicates. Statistical comparisons performed using one-way ANOVA. Asterisks indicate statistical significance between groups. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: The LNCaP and C4-2 PCa cell lines were purchased from the American Type Culture Collection (ATCC), and they provided initial authentication.

Techniques: Knockdown, Glo Assay, Luminescence Assay