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A) C4-2 cells were treated with BLU-222 (500 nM) and Tagtociclib (500 nM) for 3 days, R-loop accumulation was detected using immunofluorescence (the S9.6 antibody), and TP53BP1 was also detected. Quantification of signal intensity with SEM and significance was assessed by paired two-tailed Student’s t -test. B) CRPC cell lines (C4-2 and 22RV1) and normal prostate epithelial cells (PNT1) were treated for 4 days <t>with</t> <t>CX-5461</t> or Everolimus alone, or in combination with BLU-222 (500 nM) or Tagtociclib (500 nM), cell viability assessed and data is the mean of four biological replicates (2-3 technical replicates each) with SEM. Paired two-tailed Student’s t -test was used to evaluate statistical significance, which is indicated in comparison between CDK2 inhibitor-alone and compound of interest-alone to combination and is color-coded according to CDK2 inhibitor used. C) Colony-formation assay. Cells were either not radiated (0), first radiated and then treated after 1 day (R>C), first treated and then radiated after 1 day (C>R) or treated and radiated at the same time (CR). After 7 days, the colonies were detected (4 biological replicates with SEM). Paired two-tailed Student’s t -test was used to evaluate statistical significance and the value reported is in comparison to both radiation-alone and CDK2 inhibitor-alone (whichever is higher, is reported).
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A) C4-2 cells were treated with BLU-222 (500 nM) and Tagtociclib (500 nM) for 3 days, R-loop accumulation was detected using immunofluorescence (the S9.6 antibody), and TP53BP1 was also detected. Quantification of signal intensity with SEM and significance was assessed by paired two-tailed Student’s t -test. B) CRPC cell lines (C4-2 and 22RV1) and normal prostate epithelial cells (PNT1) were treated for 4 days <t>with</t> <t>CX-5461</t> or Everolimus alone, or in combination with BLU-222 (500 nM) or Tagtociclib (500 nM), cell viability assessed and data is the mean of four biological replicates (2-3 technical replicates each) with SEM. Paired two-tailed Student’s t -test was used to evaluate statistical significance, which is indicated in comparison between CDK2 inhibitor-alone and compound of interest-alone to combination and is color-coded according to CDK2 inhibitor used. C) Colony-formation assay. Cells were either not radiated (0), first radiated and then treated after 1 day (R>C), first treated and then radiated after 1 day (C>R) or treated and radiated at the same time (CR). After 7 days, the colonies were detected (4 biological replicates with SEM). Paired two-tailed Student’s t -test was used to evaluate statistical significance and the value reported is in comparison to both radiation-alone and CDK2 inhibitor-alone (whichever is higher, is reported).
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A) C4-2 cells were treated with BLU-222 (500 nM) and Tagtociclib (500 nM) for 3 days, R-loop accumulation was detected using immunofluorescence (the S9.6 antibody), and TP53BP1 was also detected. Quantification of signal intensity with SEM and significance was assessed by paired two-tailed Student’s t -test. B) CRPC cell lines (C4-2 and 22RV1) and normal prostate epithelial cells (PNT1) were treated for 4 days <t>with</t> <t>CX-5461</t> or Everolimus alone, or in combination with BLU-222 (500 nM) or Tagtociclib (500 nM), cell viability assessed and data is the mean of four biological replicates (2-3 technical replicates each) with SEM. Paired two-tailed Student’s t -test was used to evaluate statistical significance, which is indicated in comparison between CDK2 inhibitor-alone and compound of interest-alone to combination and is color-coded according to CDK2 inhibitor used. C) Colony-formation assay. Cells were either not radiated (0), first radiated and then treated after 1 day (R>C), first treated and then radiated after 1 day (C>R) or treated and radiated at the same time (CR). After 7 days, the colonies were detected (4 biological replicates with SEM). Paired two-tailed Student’s t -test was used to evaluate statistical significance and the value reported is in comparison to both radiation-alone and CDK2 inhibitor-alone (whichever is higher, is reported).
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A) C4-2 cells were treated with BLU-222 (500 nM) and Tagtociclib (500 nM) for 3 days, R-loop accumulation was detected using immunofluorescence (the S9.6 antibody), and TP53BP1 was also detected. Quantification of signal intensity with SEM and significance was assessed by paired two-tailed Student’s t -test. B) CRPC cell lines (C4-2 and 22RV1) and normal prostate epithelial cells (PNT1) were treated for 4 days <t>with</t> <t>CX-5461</t> or Everolimus alone, or in combination with BLU-222 (500 nM) or Tagtociclib (500 nM), cell viability assessed and data is the mean of four biological replicates (2-3 technical replicates each) with SEM. Paired two-tailed Student’s t -test was used to evaluate statistical significance, which is indicated in comparison between CDK2 inhibitor-alone and compound of interest-alone to combination and is color-coded according to CDK2 inhibitor used. C) Colony-formation assay. Cells were either not radiated (0), first radiated and then treated after 1 day (R>C), first treated and then radiated after 1 day (C>R) or treated and radiated at the same time (CR). After 7 days, the colonies were detected (4 biological replicates with SEM). Paired two-tailed Student’s t -test was used to evaluate statistical significance and the value reported is in comparison to both radiation-alone and CDK2 inhibitor-alone (whichever is higher, is reported).
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A) C4-2 cells were treated with BLU-222 (500 nM) and Tagtociclib (500 nM) for 3 days, R-loop accumulation was detected using immunofluorescence (the S9.6 antibody), and TP53BP1 was also detected. Quantification of signal intensity with SEM and significance was assessed by paired two-tailed Student’s t -test. B) CRPC cell lines (C4-2 and 22RV1) and normal prostate epithelial cells (PNT1) were treated for 4 days with CX-5461 or Everolimus alone, or in combination with BLU-222 (500 nM) or Tagtociclib (500 nM), cell viability assessed and data is the mean of four biological replicates (2-3 technical replicates each) with SEM. Paired two-tailed Student’s t -test was used to evaluate statistical significance, which is indicated in comparison between CDK2 inhibitor-alone and compound of interest-alone to combination and is color-coded according to CDK2 inhibitor used. C) Colony-formation assay. Cells were either not radiated (0), first radiated and then treated after 1 day (R>C), first treated and then radiated after 1 day (C>R) or treated and radiated at the same time (CR). After 7 days, the colonies were detected (4 biological replicates with SEM). Paired two-tailed Student’s t -test was used to evaluate statistical significance and the value reported is in comparison to both radiation-alone and CDK2 inhibitor-alone (whichever is higher, is reported).

Journal: bioRxiv

Article Title: Castration-resistant prostate cancer cells are addicted to the high activity of cyclin-dependent kinase 2

doi: 10.64898/2026.03.17.712428

Figure Lengend Snippet: A) C4-2 cells were treated with BLU-222 (500 nM) and Tagtociclib (500 nM) for 3 days, R-loop accumulation was detected using immunofluorescence (the S9.6 antibody), and TP53BP1 was also detected. Quantification of signal intensity with SEM and significance was assessed by paired two-tailed Student’s t -test. B) CRPC cell lines (C4-2 and 22RV1) and normal prostate epithelial cells (PNT1) were treated for 4 days with CX-5461 or Everolimus alone, or in combination with BLU-222 (500 nM) or Tagtociclib (500 nM), cell viability assessed and data is the mean of four biological replicates (2-3 technical replicates each) with SEM. Paired two-tailed Student’s t -test was used to evaluate statistical significance, which is indicated in comparison between CDK2 inhibitor-alone and compound of interest-alone to combination and is color-coded according to CDK2 inhibitor used. C) Colony-formation assay. Cells were either not radiated (0), first radiated and then treated after 1 day (R>C), first treated and then radiated after 1 day (C>R) or treated and radiated at the same time (CR). After 7 days, the colonies were detected (4 biological replicates with SEM). Paired two-tailed Student’s t -test was used to evaluate statistical significance and the value reported is in comparison to both radiation-alone and CDK2 inhibitor-alone (whichever is higher, is reported).

Article Snippet: BLU-222, tagtociclib, docetaxel, cabazitaxel, carboplatin, cisplatin, etoposide, olaparib, rucaparib, everolimus, CX-5461, enzalutamide and darolutamide were obtained from MedChemExpress.

Techniques: Immunofluorescence, Two Tailed Test, Comparison, Colony Assay