Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Volcano plot of RNA Pol II-interacting proteins in C4-2 cells treated with 150 nM THZ531 (CDK12/13i), 20 nM NVP-2 (CDK9i) and 500 nM YKL-5-124 (CDK7i) for 4 h. The results show that 127, 43 and 72 proteins are significantly enriched under CDK12i, CDK9i and CDK7i, respectively, and 62, 18 and 12 proteins are significantly decreased (p < 0.05 and log2FC ± 1). B Upset plots compare the distribution of proteins that are significantly lost and gained after inhibition of CDK7, CDK9, or CDK12 under the 5-EU metabolic labeling method. C The dotplot shows the GO molecular function enrichment analysis of proteins that were significantly increased after inhibition of CDK7, CDK9, and CDK12. CDK12 inhibition specifically upregulated and enriched the “Damaged DNA Binding” and “Histone Acetylation” pathways, while CDK7i and CDK9i do not. D The protein-protein interaction (PPI) network (STRING database) of the adaptive complex formed around RNA Pol II, which exhibits significant increased interaction with RNA Pol II upon CDK12 inhibition, is centered on DDB1, BRD4, PAF1, and ELOA.

Article Snippet: Antibodies used are from ThermoFisher Scientific: DDB1 (2B12D1) and PAF1 (A300-172A); Santa Cruz Biotechnology: RNA Pol II (sc-56567), GAPDH (sc-47724); Proteintech: BRD4 (28486-1-AP) and from Abcam: Actin (ab49900).

Techniques: Inhibition, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Prostate cancer cells depend on major transcription-associated cyclin-dependent kinases (CDK7, CDK9 and CDK12), whereas normal prostate cells show relative resistance to inhibitors targeting these kinases. Each cell line was cultured for 24 hours before treatment and then treated for 4 days in 50 nM YKL-5-124, 20 nM NVP-2 and 150 nM THZ531, respectively, and their cell viability was assessed using CellTiterGlo. The data is from three biological replicates, each having three technical replicates. B The results show that in the C4-2 cell line, PAF1 knockdown produced significant combined decrease in viability only with CDK12 inhibition. DDB1 knockdown produced significant combined decrease in viability with CDK12 and CDK9 inhibition, but not with CDK7 inhibition. BRD4 knockdown produced significant combined decrease in viability with CDK12, CDK9, and CDK7 inhibition. In the 22RV1 cell line, DDB1 and BRD4 knockdown produced combined decrease in viability with CDK12, CDK9, and CDK7 inhibition, but PAF1 knockdown produced significant combined lethality only with CDK12 inhibition. Cell viability after 4 days of DDB1, BRD4, and PAF1 knockdown with CDK7, CDK9, and CDK12 inhibitor treatments for the last three days. Data were obtained from three biological replicates (each containing three technical replicates), and the standard error of the mean was calculated. Student’s t -test was used to assess significance. C The knockdown of DDB1, BRD4, and PAF1 was confirmed by western blotting in castration-resistant prostate cancer cell lines C4-2 and 22RV1 (n=2).

Article Snippet: Antibodies used are from ThermoFisher Scientific: DDB1 (2B12D1) and PAF1 (A300-172A); Santa Cruz Biotechnology: RNA Pol II (sc-56567), GAPDH (sc-47724); Proteintech: BRD4 (28486-1-AP) and from Abcam: Actin (ab49900).

Techniques: Cell Culture, Knockdown, Produced, Inhibition, Western Blot

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Knockdown of PAF1 in androgen-dependent cell line LNCaP induces combined reduction in cell viability with CDK12 inhibition, but does not trigger this combined effect with CDK9 and CDK7 inhibition. Knockdown of DDB1 in LNCaP induces combinatorial anti-proliferative effects with CDK12/13, CDK9, and CDK7 inhibition. Knockdown of BRD4 in LNCaP decreased cell viability with CDK9 and CDK7 inhibition but this effect is not significant with 150 nM treatment (CDK12/13) inhibition. B Knockdown of DDB1, BRD4, and PAF1 in the cell line immortalized from normal prostate epithelia, PNT1 does not induce significant combinatorial anti-proliferative effects with CDK12/13, CDK9, and CDK7 inhibition. Data were obtained from 3 biological replicates (each biological replicate contained 3 technical replicates), and the standard error of the mean was calculated. Significance was assessed using Student’s t -test.

Article Snippet: Antibodies used are from ThermoFisher Scientific: DDB1 (2B12D1) and PAF1 (A300-172A); Santa Cruz Biotechnology: RNA Pol II (sc-56567), GAPDH (sc-47724); Proteintech: BRD4 (28486-1-AP) and from Abcam: Actin (ab49900).

Techniques: Knockdown, Inhibition

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Volcano plot of RNA Pol II-interacting proteins in C4-2 cells treated with 150 nM THZ531 (CDK12/13i), 20 nM NVP-2 (CDK9i) and 500 nM YKL-5-124 (CDK7i) for 4 h. The results show that 127, 43 and 72 proteins are significantly enriched under CDK12i, CDK9i and CDK7i, respectively, and 62, 18 and 12 proteins are significantly decreased (p < 0.05 and log2FC ± 1). B Upset plots compare the distribution of proteins that are significantly lost and gained after inhibition of CDK7, CDK9, or CDK12 under the 5-EU metabolic labeling method. C The dotplot shows the GO molecular function enrichment analysis of proteins that were significantly increased after inhibition of CDK7, CDK9, and CDK12. CDK12 inhibition specifically upregulated and enriched the “Damaged DNA Binding” and “Histone Acetylation” pathways, while CDK7i and CDK9i do not. D The protein-protein interaction (PPI) network (STRING database) of the adaptive complex formed around RNA Pol II, which exhibits significant increased interaction with RNA Pol II upon CDK12 inhibition, is centered on DDB1, BRD4, PAF1, and ELOA.

Article Snippet: Antibodies used are from ThermoFisher Scientific: DDB1 (2B12D1) and PAF1 (A300-172A); Santa Cruz Biotechnology: RNA Pol II (sc-56567), GAPDH (sc-47724); Proteintech: BRD4 (28486-1-AP) and from Abcam: Actin (ab49900).

Techniques: Inhibition, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Prostate cancer cells depend on major transcription-associated cyclin-dependent kinases (CDK7, CDK9 and CDK12), whereas normal prostate cells show relative resistance to inhibitors targeting these kinases. Each cell line was cultured for 24 hours before treatment and then treated for 4 days in 50 nM YKL-5-124, 20 nM NVP-2 and 150 nM THZ531, respectively, and their cell viability was assessed using CellTiterGlo. The data is from three biological replicates, each having three technical replicates. B The results show that in the C4-2 cell line, PAF1 knockdown produced significant combined decrease in viability only with CDK12 inhibition. DDB1 knockdown produced significant combined decrease in viability with CDK12 and CDK9 inhibition, but not with CDK7 inhibition. BRD4 knockdown produced significant combined decrease in viability with CDK12, CDK9, and CDK7 inhibition. In the 22RV1 cell line, DDB1 and BRD4 knockdown produced combined decrease in viability with CDK12, CDK9, and CDK7 inhibition, but PAF1 knockdown produced significant combined lethality only with CDK12 inhibition. Cell viability after 4 days of DDB1, BRD4, and PAF1 knockdown with CDK7, CDK9, and CDK12 inhibitor treatments for the last three days. Data were obtained from three biological replicates (each containing three technical replicates), and the standard error of the mean was calculated. Student’s t -test was used to assess significance. C The knockdown of DDB1, BRD4, and PAF1 was confirmed by western blotting in castration-resistant prostate cancer cell lines C4-2 and 22RV1 (n=2).

Article Snippet: Antibodies used are from ThermoFisher Scientific: DDB1 (2B12D1) and PAF1 (A300-172A); Santa Cruz Biotechnology: RNA Pol II (sc-56567), GAPDH (sc-47724); Proteintech: BRD4 (28486-1-AP) and from Abcam: Actin (ab49900).

Techniques: Cell Culture, Knockdown, Produced, Inhibition, Western Blot

Journal: bioRxiv

Article Title: Chromatin compaction upon CDK12 inhibition drives long gene silencing and a combinatorial lethal dependency on PAF1

doi: 10.64898/2026.01.27.701921

Figure Lengend Snippet: A Knockdown of PAF1 in androgen-dependent cell line LNCaP induces combined reduction in cell viability with CDK12 inhibition, but does not trigger this combined effect with CDK9 and CDK7 inhibition. Knockdown of DDB1 in LNCaP induces combinatorial anti-proliferative effects with CDK12/13, CDK9, and CDK7 inhibition. Knockdown of BRD4 in LNCaP decreased cell viability with CDK9 and CDK7 inhibition but this effect is not significant with 150 nM treatment (CDK12/13) inhibition. B Knockdown of DDB1, BRD4, and PAF1 in the cell line immortalized from normal prostate epithelia, PNT1 does not induce significant combinatorial anti-proliferative effects with CDK12/13, CDK9, and CDK7 inhibition. Data were obtained from 3 biological replicates (each biological replicate contained 3 technical replicates), and the standard error of the mean was calculated. Significance was assessed using Student’s t -test.

Article Snippet: Antibodies used are from ThermoFisher Scientific: DDB1 (2B12D1) and PAF1 (A300-172A); Santa Cruz Biotechnology: RNA Pol II (sc-56567), GAPDH (sc-47724); Proteintech: BRD4 (28486-1-AP) and from Abcam: Actin (ab49900).

Techniques: Knockdown, Inhibition

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) MCL clustering of STRING Chd8 protein-protein network with all proteins identified by either Chd8 antibody via either SAINTexpress or DEP at a P-value < 0.05 significance cutoff. MCL clustering was performed with the granularity parameter set to 6. Color of the outside ring indicates the annotated GO term. Inner circle color indicates if the protein was included in the more stringent 222 Chd8 interacting protein set. Network was made using default settings for a physical STRING network indicating the proteins are part of a physical complex. B) Western blotting of Co-IP validation of Chd8 protein interactions. Co-IP was performed using either the C-terminus or N- terminus Chd8 antibody, indicated by the top labels. The antibody used for western blotting is indicated to the right and the molecular weight (kDa) of the band to the left. C) Colocalization validation of protein interactions. D) Schematic of Proximity Ligation Assay (PLA) approach. PLA visualizes protein interactions via fluorescent signal amplification when targets are in close molecular proximity. E) PLA validation of Chd8 protein interactions. Nuclei were stained blue with Hoechst, with red dots indicating locations where the two target proteins are interacting. F-G) Western blotting of Chd8 Co-IP after RNase treatment. +/- indicates the presence or absence of RNase, respectively. The top labels indicate the antibody used for Co-IP. F) Co-IP of Rbm8a. G) Co-IP of Paf1.

Article Snippet: Other protein targets include MAP2 (Invitrogen, 13-1500), PAF1 (Bethyl, A300-172A), and POLD1 (Bethyl, A301-007A).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Biomarker Discovery, Molecular Weight, Proximity Ligation Assay, Amplification, Staining