Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) Schematic model of nucleolar structure (left). Immunofluorescence (IF) analysis of the indicated nucleolar marker expression in OS cells incubated with E9 (200 nM x 6 h). Scale bar, 5 μm. (B) IF analysis of H3K9me3 staining in OS cells treated as in (A). Scale bar, 10 μm, inset, 1 μm. (C) RNA-ISH analysis of 28S rRNA in OS cells treated as in (A). Scale bar, 10 μm. (D) RT-qPCR analysis of rRNA expression in OS cells treated with E9 as in A or BSJ-4-116 (500 mM x 6 h). GAPDH was used as loading control. Data represent mean ± standard deviation (S.D.) of 3 independent replicates. (E). RT-qPCR analysis of rRNA expression (right) at the regions corresponding to the schematic model of the rDNA tandem repetitive structure (left) in OS cells treated as in (D). Blue boxes represent the 47S rDNA region. H4 and H13 represent primers binding to 18S and 28S, respectively. ITS-1 (Intragenic Spacer 1); ITS-2 (Intragenic Spacer 2); 3’-ETS (3’-Externally Transcribed Spacer); IGS (intergenic spacer); Pol I TES (Pol I Transcription End Site). GAPDH was used as loading control. Data represent mean ± S.D. of 3 independent replicates. * P < 0.05, and *** P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparisons test. (F) RT-qPCR analysis of rRNA expression in 143B CDK12 dTAG cells transfected with siControl and treated with DMSO or 1 µM dTAG13 (left), transfected with siControl or siCDK13 (middle), and transfected with siCDK13 and treated with DMSO or 1 µM dTAG13 (right). dTAG13 treatment and CDK13 knockdown were performed for 2 or 4 days. GAPDH was used as loading control. Data represent mean ± S.D. of 2 independent replicates. * P < 0.05, and ** P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Next, 143B cells were seeded onto 10 cm plates and co-transfected with 1 µg of pCRIS-PITChv2-BSD R -dTAG-CDK12 and 2 µg of pX330A-CDK12/PITCh, using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027) in accordance with the manufacturer’s instructions.

Techniques: Immunofluorescence, Marker, Expressing, Incubation, Staining, Quantitative RT-PCR, Control, Standard Deviation, Binding Assay, Transfection, Knockdown

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) RT-qPCR analysis of 18S, 28S and IGS expression (at the regions indicated in the cartoon above) in OS cells treated with E9 (200 nM x 6 h). (B) RT-qPCR analysis of the indicated rRNAs in OS cells treated with E9 (200 nM) for the indicated times. Data represent mean ± S.D. of 2 independent replicates in A and B. (C) RT-qPCR analysis of 18S and H16 IGS region in OS cells incubated with BSJ-4-116 (500 nM x 6 h) or CR8 (600 nM x 6 h). Data represent mean ± S.D. of 3 independent replicates. GAPDH was used as loading control in all three assays. (D) Integrative Genomics Viewer (IGV) tracks of RNA expression in forward and reverse orientations from analysis of published RNA sequencing data sets of the indicated cell lines treated with CDK12/13 inhibitors at the following doses and durations: HEK293T: THZ531 (400 nM x 6 h); THP-1: THZ531 (200 nM x 6 h); KBM7: THZ531 (600 nM x 5 h); JURKAT: THZ531 (250 nM x 8 h); 143B: E9 (200 nM x 6 h); KELLY: 400 nM (THZ531 x 6 h). The y-axis scale indicates the range of normalized read coverage for each sample. Data set accession numbers are given in Supplemental Table 2. Dashed lines, H16 locus. (E) Northern blot analysis of RNA from OS cells treated with E9 (200 nM x 6 h) or NVP-2 (50 nM x 6 h). Equal loading across the samples is indicated by SYBR gold staining (left panels) of total RNA. The H16 transcripts were detected using radiolabeled probes targeting the forward ( P-Forward) or reverse ( P-Reverse) strands.

Article Snippet: Next, 143B cells were seeded onto 10 cm plates and co-transfected with 1 µg of pCRIS-PITChv2-BSD R -dTAG-CDK12 and 2 µg of pX330A-CDK12/PITCh, using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027) in accordance with the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Control, RNA Expression, RNA Sequencing, Northern Blot, Staining

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) Heatmap showing the differential expression levels of RNA exosome and nuclear exosome co-factors in IMR32, Kelly, MG63.3, and 143B cells treated with THZ531 (500 nM x 6 h) or E9 (200 nM x 6 h) compared to DMSO treated control. Expression levels were obtained from public RNA-seq datasets GSE113314 and GSE132233 from the GEO database. Differential expression is represented as log2(fold change), with upregulation shown in red and downregulation in blue. (B) Violin plot showing the average gene length of core RNA exosome, NEXT, PAXT and TRAMP components. (C) IF analysis of poly(A) and 28S rRNA in OS cells transfected with siRNA against ZCCHC7 or TENT4A mRNA and incubated with E9 (200 nM x 6 h). Scale bar, 5 μm, inset, 1 μm. (D) IF analysis of the same markers as in (C) in OS cells treated with E9 (200 nM x 6 h) and/or RG-7834 (10 μM x 6 h). Scale bar, 5 μm, inset, 1 μm. (E) RIP-qPCR analysis of MTREX binding to 18S, 28S, H16 and H27 rRNAs in OS cells treated with E9 (200 nM x 6 h). Data represent mean ± S.D. of 2 independent replicates. RNA enrichment was calculated as (% of input of rRNA or IGS)/(% of input of GAPDH). (F) IF analysis of 28S rRNA and MTREX in OS cells treated as in (E). Scale bar, 5 μm. (G) RIP-qPCR analysis of EXOSC2 binding to 18S, 28S, H16 and H27 rRNAs in OS cells treated as in (E). Data represent mean ± S.D. of 2 independent replicates. RNA enrichment was calculated as in (E). (H) Schematic model for pAR rings accumulation following CDK12/13 inhibition in OS cells.

Article Snippet: Next, 143B cells were seeded onto 10 cm plates and co-transfected with 1 µg of pCRIS-PITChv2-BSD R -dTAG-CDK12 and 2 µg of pX330A-CDK12/PITCh, using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Control, RNA Sequencing, Transfection, Incubation, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) Schematic model of nucleolar structure (left). Immunofluorescence (IF) analysis of the indicated nucleolar marker expression in OS cells incubated with E9 (200 nM x 6 h). Scale bar, 5 μm. (B) IF analysis of H3K9me3 staining in OS cells treated as in (A). Scale bar, 10 μm, inset, 1 μm. (C) RNA-ISH analysis of 28S rRNA in OS cells treated as in (A). Scale bar, 10 μm. (D) RT-qPCR analysis of rRNA expression in OS cells treated with E9 as in A or BSJ-4-116 (500 mM x 6 h). GAPDH was used as loading control. Data represent mean ± standard deviation (S.D.) of 3 independent replicates. (E). RT-qPCR analysis of rRNA expression (right) at the regions corresponding to the schematic model of the rDNA tandem repetitive structure (left) in OS cells treated as in (D). Blue boxes represent the 47S rDNA region. H4 and H13 represent primers binding to 18S and 28S, respectively. ITS-1 (Intragenic Spacer 1); ITS-2 (Intragenic Spacer 2); 3’-ETS (3’-Externally Transcribed Spacer); IGS (intergenic spacer); Pol I TES (Pol I Transcription End Site). GAPDH was used as loading control. Data represent mean ± S.D. of 3 independent replicates. * P < 0.05, and *** P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparisons test. (F) RT-qPCR analysis of rRNA expression in 143B CDK12 dTAG cells transfected with siControl and treated with DMSO or 1 µM dTAG13 (left), transfected with siControl or siCDK13 (middle), and transfected with siCDK13 and treated with DMSO or 1 µM dTAG13 (right). dTAG13 treatment and CDK13 knockdown were performed for 2 or 4 days. GAPDH was used as loading control. Data represent mean ± S.D. of 2 independent replicates. * P < 0.05, and ** P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Next, 143B cells were seeded onto 10 cm plates and co-transfected with 1 µg of pCRIS-PITChv2-BSD R -dTAG-CDK12 and 2 µg of pX330A-CDK12/PITCh, using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027) in accordance with the manufacturer’s instructions.

Techniques: Immunofluorescence, Marker, Expressing, Incubation, Staining, Quantitative RT-PCR, Control, Standard Deviation, Binding Assay, Transfection, Knockdown

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) RT-qPCR analysis of 18S, 28S and IGS expression (at the regions indicated in the cartoon above) in OS cells treated with E9 (200 nM x 6 h). (B) RT-qPCR analysis of the indicated rRNAs in OS cells treated with E9 (200 nM) for the indicated times. Data represent mean ± S.D. of 2 independent replicates in A and B. (C) RT-qPCR analysis of 18S and H16 IGS region in OS cells incubated with BSJ-4-116 (500 nM x 6 h) or CR8 (600 nM x 6 h). Data represent mean ± S.D. of 3 independent replicates. GAPDH was used as loading control in all three assays. (D) Integrative Genomics Viewer (IGV) tracks of RNA expression in forward and reverse orientations from analysis of published RNA sequencing data sets of the indicated cell lines treated with CDK12/13 inhibitors at the following doses and durations: HEK293T: THZ531 (400 nM x 6 h); THP-1: THZ531 (200 nM x 6 h); KBM7: THZ531 (600 nM x 5 h); JURKAT: THZ531 (250 nM x 8 h); 143B: E9 (200 nM x 6 h); KELLY: 400 nM (THZ531 x 6 h). The y-axis scale indicates the range of normalized read coverage for each sample. Data set accession numbers are given in Supplemental Table 2. Dashed lines, H16 locus. (E) Northern blot analysis of RNA from OS cells treated with E9 (200 nM x 6 h) or NVP-2 (50 nM x 6 h). Equal loading across the samples is indicated by SYBR gold staining (left panels) of total RNA. The H16 transcripts were detected using radiolabeled probes targeting the forward ( P-Forward) or reverse ( P-Reverse) strands.

Article Snippet: Next, 143B cells were seeded onto 10 cm plates and co-transfected with 1 µg of pCRIS-PITChv2-BSD R -dTAG-CDK12 and 2 µg of pX330A-CDK12/PITCh, using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027) in accordance with the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Control, RNA Expression, RNA Sequencing, Northern Blot, Staining

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) Heatmap showing the differential expression levels of RNA exosome and nuclear exosome co-factors in IMR32, Kelly, MG63.3, and 143B cells treated with THZ531 (500 nM x 6 h) or E9 (200 nM x 6 h) compared to DMSO treated control. Expression levels were obtained from public RNA-seq datasets GSE113314 and GSE132233 from the GEO database. Differential expression is represented as log2(fold change), with upregulation shown in red and downregulation in blue. (B) Violin plot showing the average gene length of core RNA exosome, NEXT, PAXT and TRAMP components. (C) IF analysis of poly(A) and 28S rRNA in OS cells transfected with siRNA against ZCCHC7 or TENT4A mRNA and incubated with E9 (200 nM x 6 h). Scale bar, 5 μm, inset, 1 μm. (D) IF analysis of the same markers as in (C) in OS cells treated with E9 (200 nM x 6 h) and/or RG-7834 (10 μM x 6 h). Scale bar, 5 μm, inset, 1 μm. (E) RIP-qPCR analysis of MTREX binding to 18S, 28S, H16 and H27 rRNAs in OS cells treated with E9 (200 nM x 6 h). Data represent mean ± S.D. of 2 independent replicates. RNA enrichment was calculated as (% of input of rRNA or IGS)/(% of input of GAPDH). (F) IF analysis of 28S rRNA and MTREX in OS cells treated as in (E). Scale bar, 5 μm. (G) RIP-qPCR analysis of EXOSC2 binding to 18S, 28S, H16 and H27 rRNAs in OS cells treated as in (E). Data represent mean ± S.D. of 2 independent replicates. RNA enrichment was calculated as in (E). (H) Schematic model for pAR rings accumulation following CDK12/13 inhibition in OS cells.

Article Snippet: Next, 143B cells were seeded onto 10 cm plates and co-transfected with 1 µg of pCRIS-PITChv2-BSD R -dTAG-CDK12 and 2 µg of pX330A-CDK12/PITCh, using Lipofectamine 2000 (Thermo Fisher Scientific, 11668027) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Control, RNA Sequencing, Transfection, Incubation, Binding Assay, Inhibition

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) Schematic model of nucleolar structure (left). Immunofluorescence (IF) analysis of the indicated nucleolar marker expression in OS cells incubated with E9 (200 nM x 6 h). Scale bar, 5 μm. (B) IF analysis of H3K9me3 staining in OS cells treated as in (A). Scale bar, 10 μm, inset, 1 μm. (C) RNA-ISH analysis of 28S rRNA in OS cells treated as in (A). Scale bar, 10 μm. (D) RT-qPCR analysis of rRNA expression in OS cells treated with E9 as in A or BSJ-4-116 (500 mM x 6 h). GAPDH was used as loading control. Data represent mean ± standard deviation (S.D.) of 3 independent replicates. (E). RT-qPCR analysis of rRNA expression (right) at the regions corresponding to the schematic model of the rDNA tandem repetitive structure (left) in OS cells treated as in (D). Blue boxes represent the 47S rDNA region. H4 and H13 represent primers binding to 18S and 28S, respectively. ITS-1 (Intragenic Spacer 1); ITS-2 (Intragenic Spacer 2); 3’-ETS (3’-Externally Transcribed Spacer); IGS (intergenic spacer); Pol I TES (Pol I Transcription End Site). GAPDH was used as loading control. Data represent mean ± S.D. of 3 independent replicates. * P < 0.05, and *** P < 0.0001, one-way ANOVA followed by Dunnett’s multiple comparisons test. (F) RT-qPCR analysis of rRNA expression in 143B CDK12 dTAG cells transfected with siControl and treated with DMSO or 1 µM dTAG13 (left), transfected with siControl or siCDK13 (middle), and transfected with siCDK13 and treated with DMSO or 1 µM dTAG13 (right). dTAG13 treatment and CDK13 knockdown were performed for 2 or 4 days. GAPDH was used as loading control. Data represent mean ± S.D. of 2 independent replicates. * P < 0.05, and ** P < 0.001, one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: The BSD R -P2A-2xHA-FKBP12 F36V -linker cassette, generated with flanking sequences homologous to the CDK12 locus, was assembled into the pCRIS-PITChv2 backbone using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), following the manufacturer’s protocol.

Techniques: Immunofluorescence, Marker, Expressing, Incubation, Staining, Quantitative RT-PCR, Control, Standard Deviation, Binding Assay, Transfection, Knockdown

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) RT-qPCR analysis of 18S, 28S and IGS expression (at the regions indicated in the cartoon above) in OS cells treated with E9 (200 nM x 6 h). (B) RT-qPCR analysis of the indicated rRNAs in OS cells treated with E9 (200 nM) for the indicated times. Data represent mean ± S.D. of 2 independent replicates in A and B. (C) RT-qPCR analysis of 18S and H16 IGS region in OS cells incubated with BSJ-4-116 (500 nM x 6 h) or CR8 (600 nM x 6 h). Data represent mean ± S.D. of 3 independent replicates. GAPDH was used as loading control in all three assays. (D) Integrative Genomics Viewer (IGV) tracks of RNA expression in forward and reverse orientations from analysis of published RNA sequencing data sets of the indicated cell lines treated with CDK12/13 inhibitors at the following doses and durations: HEK293T: THZ531 (400 nM x 6 h); THP-1: THZ531 (200 nM x 6 h); KBM7: THZ531 (600 nM x 5 h); JURKAT: THZ531 (250 nM x 8 h); 143B: E9 (200 nM x 6 h); KELLY: 400 nM (THZ531 x 6 h). The y-axis scale indicates the range of normalized read coverage for each sample. Data set accession numbers are given in Supplemental Table 2. Dashed lines, H16 locus. (E) Northern blot analysis of RNA from OS cells treated with E9 (200 nM x 6 h) or NVP-2 (50 nM x 6 h). Equal loading across the samples is indicated by SYBR gold staining (left panels) of total RNA. The H16 transcripts were detected using radiolabeled probes targeting the forward ( P-Forward) or reverse ( P-Reverse) strands.

Article Snippet: The BSD R -P2A-2xHA-FKBP12 F36V -linker cassette, generated with flanking sequences homologous to the CDK12 locus, was assembled into the pCRIS-PITChv2 backbone using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), following the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Incubation, Control, RNA Expression, RNA Sequencing, Northern Blot, Staining

Journal: bioRxiv

Article Title: CDK12/13 inhibition disrupts nucleolar morphology and promotes aberrant expression of IGS transcripts

doi: 10.1101/2025.03.01.640972

Figure Lengend Snippet: (A) Heatmap showing the differential expression levels of RNA exosome and nuclear exosome co-factors in IMR32, Kelly, MG63.3, and 143B cells treated with THZ531 (500 nM x 6 h) or E9 (200 nM x 6 h) compared to DMSO treated control. Expression levels were obtained from public RNA-seq datasets GSE113314 and GSE132233 from the GEO database. Differential expression is represented as log2(fold change), with upregulation shown in red and downregulation in blue. (B) Violin plot showing the average gene length of core RNA exosome, NEXT, PAXT and TRAMP components. (C) IF analysis of poly(A) and 28S rRNA in OS cells transfected with siRNA against ZCCHC7 or TENT4A mRNA and incubated with E9 (200 nM x 6 h). Scale bar, 5 μm, inset, 1 μm. (D) IF analysis of the same markers as in (C) in OS cells treated with E9 (200 nM x 6 h) and/or RG-7834 (10 μM x 6 h). Scale bar, 5 μm, inset, 1 μm. (E) RIP-qPCR analysis of MTREX binding to 18S, 28S, H16 and H27 rRNAs in OS cells treated with E9 (200 nM x 6 h). Data represent mean ± S.D. of 2 independent replicates. RNA enrichment was calculated as (% of input of rRNA or IGS)/(% of input of GAPDH). (F) IF analysis of 28S rRNA and MTREX in OS cells treated as in (E). Scale bar, 5 μm. (G) RIP-qPCR analysis of EXOSC2 binding to 18S, 28S, H16 and H27 rRNAs in OS cells treated as in (E). Data represent mean ± S.D. of 2 independent replicates. RNA enrichment was calculated as in (E). (H) Schematic model for pAR rings accumulation following CDK12/13 inhibition in OS cells.

Article Snippet: The BSD R -P2A-2xHA-FKBP12 F36V -linker cassette, generated with flanking sequences homologous to the CDK12 locus, was assembled into the pCRIS-PITChv2 backbone using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), following the manufacturer’s protocol.

Techniques: Expressing, Control, RNA Sequencing, Transfection, Incubation, Binding Assay, Inhibition