cdk7  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Gene expression and protein analysis of human FLC samples (A) mRNA expression of various known super-enhancer-driven genes in FLC were assessed in human tumor samples ( n = 35) and paired normal liver samples ( n = 10) ( t test, ∗∗ p < 0.0001). (B) Individual patient tumor samples with matched normal liver (FCF 82, 83, and 106) were evaluated for phosphorylated RPB-1 (Ser 2, 5, and 7) and CDK7. Fibrolamellar cancer was confirmed by demonstrating presence (tumor) and absence (normal) of the DNAJ-PKAc oncoprotein. (C) A phosphorylation index was calculated for each sample and phosphorylation level is presented as Phosphorylation index (Tumor)/Phosphorylation index (Adjacent) for RPB-1 (Ser 2, 5, and 7) and CDK7. Seven to nine sets of samples are shown from FLC tumor and adjacent normal tissue samples (including samples from ). (D) The data suggest that DNAJ-PKAc is correlated with heightened CDK7 activity (dotted green line). CDK7 forms a trimeric complex with cyclin H and MAT1 to phosphorylate serine-5 (preferentially) and serine-7 residues of a 52 heptad repeat in RNA polymerase II. Similarly, CDK9 dimerizes with cyclin T to preferentially phosphorylate serine-2 residues. SY-5609 and YKL-5-124 inhibit CDK7 activity; similarly VIP-152 and NVP-2 inhibit CDK9 activity. [Image created with Biorender.com ].

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 is a novel therapeutic target in DNAJB1-PRKACA-expressing cells (A) To assess for CDK7 effect on cell viability, HepG2 cells and H33 cells underwent 48-h drug treatment with either SY5609 (1 pM–5 μM) or YKL-5-124 (100 pM–10 μM). Percent viability was determined by normalizing to control (DMSO treated). To confirm the findings in the DNAJB1-PRKACA -expressing H33 cells, a separate clone (H12) was tested with the same drugs over the same dose range. In each figure, the LC 50 (IC 50 ) is represented by the straight line. (B) HepG2 cells and H33 cells were synchronized and treated with either DMSO or SY5609 (1 μm) for 24 h. Percent of cells in G0/G1, S, and G2/M were determined by flow cytometry. Shown are four biological replicates per cell line per treatment ( t test, ∗ p = 0.001, ∗∗ p < 0.0001). (C) HepG2 cells and H33 cells were treated with DMSO (control) or increasing doses of SY5609 for 24 h and caspase 3/7 activity measured. To confirm the increased apoptotic activity in the H33 cells, a separate clone (H12) was utilized. ( t test ∗ p < 0.0001, for H33 vs. HepG2 and H12 vs. HepG2). (D) To validate the results, PARP and cleaved-PARP (marker for apoptosis) protein were evaluated in HepG2 and H33 cells, using two separate antibodies that either recognize both PARP and cleaved-PARP (top bands) or only cleaved-PARP alone (bottom band). (E) Primary human hepatocytes (PHHs) isolated fresh from human donor liver transplant specimens and H33 cells were treated with DMSO, SY5609 100 nM, or SY5609 1 μM for 24 and 48 h. The percent of viable cells was determined by normalizing to DMSO control. There were four biological replicates per group per time/treatment dose ( t test ∗ p = 0.02, ∗∗ p = 0.01, ∗∗∗ p < 0.0001). (F) PHHs and H33 cells were treated with SY5609 (100 pM–5 μM) for 48, 72, or 120 h and percent viability assessed, normalized to DMSO control. The LC 50 (IC 50 ) is demarcated by the solid line. There were four biological replicates for each drug dose at each time point for each line (PHH and H33).

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Expressing, Control, Flow Cytometry, Activity Assay, Marker, Isolation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 inhibition is lethal in human FLC (A) An FLC cell line derived from human FLC (FLC-H) was grown for six days (control) or treated with DMSO (Control DMSO) versus SY5609 at two separate doses (500 nM and 1 μM). Viable cells were quantified at day zero and day six. Additionally, the day 6 percent survival compared to controls was calculated. Shown are two separate experiments with three biological replicates per experiment ( t test ∗ p < 0.01, ∗∗ p < 0.001 versus control and control DMSO). (B) In a separate experiment, an FLC cell line was derived from a patient FLC liver tumor (FLC1025), and another discrete cell line was derived from patient metastatic FLC tumor implants (FLCMet). Each line was treated with SY5609 (10 nM–10 μM). Percent survival was determined normalized to DMSO control, with three biological replicates per dose. Shown is the dose response with curve (and 95% CI) and LC 50 (IC 50 ) demonstrated by the straight line (LC 50 FLC1025 ∼300 nM, LC 50 FLCMet ∼20 nM). (C) Human tissue slices derived from a patient with FLC (FLC217) were treated with DMSO or SY5609 (500 nM), and percent viability determined compared to DMSO control (∗∗ p = 0.003). (D) In a separate experiment, tissue slices derived from human FLC grown in a patient derived xenograft (PDX) model were treated with DMSO, SY5609 (100 nM, 500 nM, and 1 μM) and staurosporine (STS) 500 nM (positive control). There were 3–4 biological replicates per group ( t test ∗ p < 0.01, ∗∗ p < 0.001).

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Inhibition, Derivative Assay, Control, Positive Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination between CDK7 and CDK9 inhibition in vitro (A) DNAJB1-PRKACA expressing H33 cells were treated with SY-5609 alone, VIP-152 alone, or in combination for 24 h. Protein was isolated and western blot performed for measurement and quantification of phosphorylated RPB-1 (Ser 2, 5, and 7) ( n = 3 biological replicates per group, two representative western blots are shown, t test, ∗ p < 0.05). (B) Expression of SLC16A14 and LINC00473 was determined in H33 cells treated with SY-5609 alone (blue), VIP-152 alone (purple), or in combination (green). Statistical significance is denoted as compared to control ( t test, ∗∗ p < 0.01, ∗ p < 0.05). (C and D) Synergistic response to combination therapy of SY-5609 and VIP-152 in H33 cells was determined with potent reduction in percent survival (normalized to DMSO control). Depicted is a dose-response curve which demonstrates shift of the curve to the left for SY-5609 with increasing concentration of VIP-152 up to a dose of 30 nM VIP-152. The drug combination showed strong synergy using all metrics including HSA (mean 18.73, p = 6.31e-5), Bliss (mean 14.55, p = 3.46e-4), Loewe (mean 15.48, p = 1.98e-4), and ZIP (mean 13.54, p = 6.04e-4). The strongest synergistic doses occurred at 30 nM SY-5609 + 30 nM VIP152 (synergy score ∼38) and 10 nM SY-5609 + 30 nM VIP152 (synergy score ∼37).

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Inhibition, In Vitro, Expressing, Isolation, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 and CDK9 inhibition in an organoid model (A) Tissue from a patient with FLC (FLC4-PDX) was propagated and developed into a patient-derived cancer organoid model, FLC4 (organoid). Western blot shows the presence of DNAJ-PKAc oncoprotein and phosphorylated RPB-1 (Ser 2, 5, and 7) in the organoid compared to immortalized human hepatocytes (IHH, n = 4) which displays only native PKAc and low levels of phosphorylated RPB-1. Phosphorylated RPB-1 (Ser 2, 5, and 7) was measured and quantified. Statistical significance was calculated ( t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01). (B) RNA was isolated from organoids treated with SY-5609 (1 nM, 3 nM, 10 nM, and 30 nM) and VIP-152 (30 nM, 100 nM, 300 nM, and 500 nM) and SLC16A14 and LINC00473 expression was determined by qPCR relative to DMSO control. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Inhibition, Derivative Assay, Western Blot, Isolation, Expressing, Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination of CDK7 and CDK9 inhibition in an organoid model (A) FLC organoids were treated with SY-5609 alone, VIP-152 alone or in combination and western blot was performed showing phosphorylated RPB-1 (Ser 2, 5, and 7). Protein levels were measured and quantified. (B) FLC organoids were treated with SY-509 alone and in combination with VIP-152 at various doses which showed a dose-dependent decrease in cell survival as compared to DMSO. (C) RNA was isolated from FLC organoids treated with combination SY-5609 and VIP-152 for 96 h (1nM/10 nM, 10nM/10 nM, and 100nM/100 nM) and expression of SLC16A14 and LINC00473 was determined. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: CDK7 , Cell Signaling Technology , Cell Signaling Technology #2916; RRID: AB_2077142.

Techniques: Inhibition, Western Blot, Isolation, Expressing

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Gene expression and protein analysis of human FLC samples (A) mRNA expression of various known super-enhancer-driven genes in FLC were assessed in human tumor samples ( n = 35) and paired normal liver samples ( n = 10) ( t test, ∗∗ p < 0.0001). (B) Individual patient tumor samples with matched normal liver (FCF 82, 83, and 106) were evaluated for phosphorylated RPB-1 (Ser 2, 5, and 7) and CDK7. Fibrolamellar cancer was confirmed by demonstrating presence (tumor) and absence (normal) of the DNAJ-PKAc oncoprotein. (C) A phosphorylation index was calculated for each sample and phosphorylation level is presented as Phosphorylation index (Tumor)/Phosphorylation index (Adjacent) for RPB-1 (Ser 2, 5, and 7) and CDK7. Seven to nine sets of samples are shown from FLC tumor and adjacent normal tissue samples (including samples from ). (D) The data suggest that DNAJ-PKAc is correlated with heightened CDK7 activity (dotted green line). CDK7 forms a trimeric complex with cyclin H and MAT1 to phosphorylate serine-5 (preferentially) and serine-7 residues of a 52 heptad repeat in RNA polymerase II. Similarly, CDK9 dimerizes with cyclin T to preferentially phosphorylate serine-2 residues. SY-5609 and YKL-5-124 inhibit CDK7 activity; similarly VIP-152 and NVP-2 inhibit CDK9 activity. [Image created with Biorender.com ].

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Gene Expression, Expressing, Phospho-proteomics, Activity Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 is a novel therapeutic target in DNAJB1-PRKACA-expressing cells (A) To assess for CDK7 effect on cell viability, HepG2 cells and H33 cells underwent 48-h drug treatment with either SY5609 (1 pM–5 μM) or YKL-5-124 (100 pM–10 μM). Percent viability was determined by normalizing to control (DMSO treated). To confirm the findings in the DNAJB1-PRKACA -expressing H33 cells, a separate clone (H12) was tested with the same drugs over the same dose range. In each figure, the LC 50 (IC 50 ) is represented by the straight line. (B) HepG2 cells and H33 cells were synchronized and treated with either DMSO or SY5609 (1 μm) for 24 h. Percent of cells in G0/G1, S, and G2/M were determined by flow cytometry. Shown are four biological replicates per cell line per treatment ( t test, ∗ p = 0.001, ∗∗ p < 0.0001). (C) HepG2 cells and H33 cells were treated with DMSO (control) or increasing doses of SY5609 for 24 h and caspase 3/7 activity measured. To confirm the increased apoptotic activity in the H33 cells, a separate clone (H12) was utilized. ( t test ∗ p < 0.0001, for H33 vs. HepG2 and H12 vs. HepG2). (D) To validate the results, PARP and cleaved-PARP (marker for apoptosis) protein were evaluated in HepG2 and H33 cells, using two separate antibodies that either recognize both PARP and cleaved-PARP (top bands) or only cleaved-PARP alone (bottom band). (E) Primary human hepatocytes (PHHs) isolated fresh from human donor liver transplant specimens and H33 cells were treated with DMSO, SY5609 100 nM, or SY5609 1 μM for 24 and 48 h. The percent of viable cells was determined by normalizing to DMSO control. There were four biological replicates per group per time/treatment dose ( t test ∗ p = 0.02, ∗∗ p = 0.01, ∗∗∗ p < 0.0001). (F) PHHs and H33 cells were treated with SY5609 (100 pM–5 μM) for 48, 72, or 120 h and percent viability assessed, normalized to DMSO control. The LC 50 (IC 50 ) is demarcated by the solid line. There were four biological replicates for each drug dose at each time point for each line (PHH and H33).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Expressing, Control, Flow Cytometry, Activity Assay, Marker, Isolation

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 inhibition is lethal in human FLC (A) An FLC cell line derived from human FLC (FLC-H) was grown for six days (control) or treated with DMSO (Control DMSO) versus SY5609 at two separate doses (500 nM and 1 μM). Viable cells were quantified at day zero and day six. Additionally, the day 6 percent survival compared to controls was calculated. Shown are two separate experiments with three biological replicates per experiment ( t test ∗ p < 0.01, ∗∗ p < 0.001 versus control and control DMSO). (B) In a separate experiment, an FLC cell line was derived from a patient FLC liver tumor (FLC1025), and another discrete cell line was derived from patient metastatic FLC tumor implants (FLCMet). Each line was treated with SY5609 (10 nM–10 μM). Percent survival was determined normalized to DMSO control, with three biological replicates per dose. Shown is the dose response with curve (and 95% CI) and LC 50 (IC 50 ) demonstrated by the straight line (LC 50 FLC1025 ∼300 nM, LC 50 FLCMet ∼20 nM). (C) Human tissue slices derived from a patient with FLC (FLC217) were treated with DMSO or SY5609 (500 nM), and percent viability determined compared to DMSO control (∗∗ p = 0.003). (D) In a separate experiment, tissue slices derived from human FLC grown in a patient derived xenograft (PDX) model were treated with DMSO, SY5609 (100 nM, 500 nM, and 1 μM) and staurosporine (STS) 500 nM (positive control). There were 3–4 biological replicates per group ( t test ∗ p < 0.01, ∗∗ p < 0.001).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Control, Positive Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination between CDK7 and CDK9 inhibition in vitro (A) DNAJB1-PRKACA expressing H33 cells were treated with SY-5609 alone, VIP-152 alone, or in combination for 24 h. Protein was isolated and western blot performed for measurement and quantification of phosphorylated RPB-1 (Ser 2, 5, and 7) ( n = 3 biological replicates per group, two representative western blots are shown, t test, ∗ p < 0.05). (B) Expression of SLC16A14 and LINC00473 was determined in H33 cells treated with SY-5609 alone (blue), VIP-152 alone (purple), or in combination (green). Statistical significance is denoted as compared to control ( t test, ∗∗ p < 0.01, ∗ p < 0.05). (C and D) Synergistic response to combination therapy of SY-5609 and VIP-152 in H33 cells was determined with potent reduction in percent survival (normalized to DMSO control). Depicted is a dose-response curve which demonstrates shift of the curve to the left for SY-5609 with increasing concentration of VIP-152 up to a dose of 30 nM VIP-152. The drug combination showed strong synergy using all metrics including HSA (mean 18.73, p = 6.31e-5), Bliss (mean 14.55, p = 3.46e-4), Loewe (mean 15.48, p = 1.98e-4), and ZIP (mean 13.54, p = 6.04e-4). The strongest synergistic doses occurred at 30 nM SY-5609 + 30 nM VIP152 (synergy score ∼38) and 10 nM SY-5609 + 30 nM VIP152 (synergy score ∼37).

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, In Vitro, Expressing, Isolation, Western Blot, Control, Concentration Assay

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 and CDK9 inhibition in an organoid model (A) Tissue from a patient with FLC (FLC4-PDX) was propagated and developed into a patient-derived cancer organoid model, FLC4 (organoid). Western blot shows the presence of DNAJ-PKAc oncoprotein and phosphorylated RPB-1 (Ser 2, 5, and 7) in the organoid compared to immortalized human hepatocytes (IHH, n = 4) which displays only native PKAc and low levels of phosphorylated RPB-1. Phosphorylated RPB-1 (Ser 2, 5, and 7) was measured and quantified. Statistical significance was calculated ( t test, ∗∗∗ p < 0.001, ∗∗ p < 0.01). (B) RNA was isolated from organoids treated with SY-5609 (1 nM, 3 nM, 10 nM, and 30 nM) and VIP-152 (30 nM, 100 nM, 300 nM, and 500 nM) and SLC16A14 and LINC00473 expression was determined by qPCR relative to DMSO control. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Derivative Assay, Western Blot, Isolation, Expressing, Control

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: Synergistic combination of CDK7 and CDK9 inhibition in an organoid model (A) FLC organoids were treated with SY-5609 alone, VIP-152 alone or in combination and western blot was performed showing phosphorylated RPB-1 (Ser 2, 5, and 7). Protein levels were measured and quantified. (B) FLC organoids were treated with SY-509 alone and in combination with VIP-152 at various doses which showed a dose-dependent decrease in cell survival as compared to DMSO. (C) RNA was isolated from FLC organoids treated with combination SY-5609 and VIP-152 for 96 h (1nM/10 nM, 10nM/10 nM, and 100nM/100 nM) and expression of SLC16A14 and LINC00473 was determined. Due to the limited availability of organoid tissue available only one replicate is represented per dose.

Article Snippet: Membranes were incubated in 10% w/v BSA blocking buffer (Thermo Fisher, Waltham, MA) at room temperature for 1 hour and hybridized with primary antibody (PKA(c): 610981 (BD Biosciences, Franklin Lakes, NJ), CDK2: #2546 (Cell Signaling Technology, Danvers, MA), phospho-CDK2 (Thr160): #2561 (Cell Signaling Technology, Danvers, MA), CDK7: #2916 (Cell Signaling Technology, Danvers, MA), phospho-CDK7 (T170): ab155976 (Abcam, Cambrige, MA), RNA polymerase II RPB-1: RPB-1 NTD; #14958, Phospho-CTD (Ser2); #13499, Phospho-CTD (Ser5); #13523, Phospho-CTD (Ser7); #13780 (Cell Signaling Technology, Danvers, MA), SCG2; PA5-115018 (Thermo Fisher, Waltham, MA), PARP; #9542 (Cell Signaling Technology, Danvers, MA), Cleaved PARP (Asp214); #5625 (Cell Signaling Technology, Danvers, MA), β-Actin: #4967 (Cell Signaling Technology, Danvers, MA), MAB8929 (R&D Systems, Minneapolis, MN) at 4°C overnight.

Techniques: Inhibition, Western Blot, Isolation, Expressing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. In theory, acute inhibition of transcriptional CDKs, particularly those involved in phosphorylating RPB1 CTD Ser2 phosphorylation, is expected to globally suppress gene expression, predominantly affecting mRNAs with short half-lives. B. Volcano plots of gene expression derived from 4-6 hours treatment of CDK7 inhibitor THZ1 (250 nM) in ovarian cancer cell line Kuramochi , CDK9 inhibitor HH1 (10 µM) in a cell line (YB5) derived from the SW48 colon cancer cell line , or CDK12 inhibitor SR-4835 (90 nM) in triple-negative breast cancer line MDA-MB-231 . RNA-seq data were downloaded from the Gene Expression Omnibus (GEO) and analyzed. C. A volcano plot of nascent RNA expression from neuroblastoma cells (IMR32) treated with 400 nM THZ531 for 2 hours . Note that the sequencing involved 4-thiouridine-pulse labeling and included RNA spike-in control. D. (Top) selection of TCGA ovarian serous adenocarcinoma samples with low or high expression of the indicated CDK genes (the top and bottom 5% in terms of CDK7, CDK9 or CDK12 mRNA expression in ovarian cancer samples with expression data; n = 17 each group). (Bottom) volcano plots of differential gene expression in tumors with low expressing CDKs compared to those with high expressing CDKs. For all volcano plots, genes significantly upregulated or downregulated (absolute log 2 fold change (FC) ≥ 1, p < 0.1) are colored in red and blue, respectively.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Inhibition, Phospho-proteomics, Gene Expression, Derivative Assay, RNA Sequencing, RNA Expression, Sequencing, Labeling, Control, Selection, Expressing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. HCC1954 cells were treated with THZ531 or NVP-2 for 4 hours, after which whole cell lysates were harvested for fluorescent immunoblotting. The molecular weights of the fluorescent protein markers and clone identities for monoclonal antibodies are indicated. Merged images show signals from two primary antibodies raised in different species. B. Volcano plots of gene expression. HCC1954 cells were treated for 4 h with THZ531 (200 nM), or NVP-2 (200 nM), or vehicle control. Total RNA was extracted and subjected to library construction and deep sequencing. Dotted lines in the volcano plots (left) indicate the thresholds used for log-transformed fold change (1, -1). The Tukey box plots (right) illustrate the comparison between the magnitudes of change in significantly up- or down-regulated genes. **** p < 0.0001; ns, not significant (Mann-Whitney test). C. Analysis of gene size among groups of genes impacted by CDK12 or 9 inhibition. **** p < 0.0001; ns, not significant (Mann-Whitney test). D. Traces of RNA-seq coverage over the gene BLM. Note that CDK12 inhibition decreases coverage over exons, but concomitantly produces reads at positions known to harbor intronic polyadenylation. The boxed regions are also displayed with re-scaled y-axis (bottom). E. Metagene profile plots of genes that are commonly downregulated in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size. Note that CDK12 inhibition is uniquely associated with a size-dependent elongation defect. F. Overrepresentation analysis of significantly downregulated genes in HCC1954 cells that were treated as indicated in the x-axis. Gene sets in the KEGG and Reactome pathway databases were evaluated. Dot color denotes the statistical significance of gene set enrichment within the group of downregulated genes (-log q) and dot size denotes the fraction of downregulated genes corresponding to that gene set. G. Summary plot of normalized enrichment scores (NES) for gene sets that are significantly downregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote gene sets implicated in the DNA damage response. H. Venn diagram for gene sets that are downregulated by CDK12, CDK9, and CDK7 inhibition. Note that gene sets involved in the DNA damage response are specific to CDK12 inhibition. The right bottom illustrates metagene profile plots of genes in the gene set Reactome_ Homologous DNA Pairing and Strand Exchange in HCC1954 cells treated with THZ531, NVP-2, or THZ1 and that have been segmented by gene size.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Western Blot, Bioprocessing, Gene Expression, Control, Sequencing, Transformation Assay, Comparison, MANN-WHITNEY, Inhibition, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. A volcano plot of gene expression in SKBR3 cells treated with THZ531 (200 nM, 4 h) compared with vehicle control (0.04% DMSO, v/v). Blue and red dots denote individual genes with significant change in expression (fold of change > 1, q < 0.1). B. (Left) A Tukey box plot indicates that, for genes significantly altered in expression by THZ531 treatment in SKBR3 cells, the magnitude of upregulation is even greater than gene downregulation (**p < 0.01, Mann-Whitney test). (Right) Comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (****p < 0.0001, Mann-Whitney test). C. HCC1954 cells were treated with vehicle control, 200 nM THZ531 or THZ1 for 4 h, followed by lysis with 1x SDS sample buffer and fluorescent immunoblotting. Note that THZ1 treatment caused a complete electrophoretic mobility shift of total RBP1, indicating a substantial loss of CTD phosphorylation. Note that THZ1 also targets CDK12 and CDK13, and thus lacks a desired selectivity. THZ1 was chosen for the current study, instead of a more selective version of CDK7 inhibitor YKL-5-124, primarily because YKL-5-125 does not have any effect on CTD phosphorylation (Olson et al., 2019). D. A volcano plot of gene expression in HCC1954 cells treated with THZ1 (200 nM, 4 h) compared with vehicle control. Blue and red dots are genes with significant change in expression (fold of change > 1, q < 0.1). E. (Left) A Tukey box plot indicating that, for genes with expression significantly altered by THZ1 treatment in HCC1954 cells, the magnitude of downregulation is greater than gene upregulation (****p < 0.01). (Right) A comparison of gene size among groups of genes that differentially respond to CDK12 inhibition (n.s., not significant; Mann-Whitney test). F. Fluorescent immunoblotting of total cell lysates from HCC1954 cells treated with indicated doses of THZ531 for 4 or 24 hours. Note that cells treated with THZ531 for 24 hours demonstrate a reduced protein abundance of ATM, BRCA1, and EGFR, all of which are encoded by large genes (>100 kb). G. (Top) A schematic depicting mRNA with proximal or distal polyA. (Bottom) The percentage of mRNA in each treatment group (HCC1954) showing positive (proximal polyA) or negative (distal polyA) change of proximal polyadenylation site usage (△PPAU). H. Traces of RNA Seq coverage over the MAP3K9 gene. Note that CDK12 inhibition in HCC1954 cells abolishes reads corresponding to a distal polyA site (boxed) while exerting no effect on coverage over the exons.

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: Gene Expression, Control, Expressing, MANN-WHITNEY, Comparison, Inhibition, Lysis, Western Blot, Electrophoretic Mobility Shift Assay, Phospho-proteomics, Quantitative Proteomics, RNA Sequencing

Journal: bioRxiv

Article Title: Dual Modes of Gene Regulation by CDK12

doi: 10.1101/2025.09.22.677923

Figure Lengend Snippet: A. (Left) Summary plot of normalized enrichment scores (NES) for gene sets that are significantly upregulated in HCC1954 cells treated with CDK12 inhibitor (nominal p-value < 0.05). Blue dots denote MYC signature. (Right) GSEA plot of Hallmark_MYC Targets V1 for genes altered by THZ531 (200 nM, 4h) in HCC1954 (top) and SKBR3 (bottom) cells. Normalized enrichment score (NES) and p values are indicated. B. Traces of RNA-seq coverage over MYC in cells that were treated as indicated. Enlarged views for traces of CDK9 and CDK7 inhibition are shown as inserts with the y-axis scaled down. C. HCC1954 cells were treated as indicated for 4 h followed by total RNA extraction and reverse transcription. Quantitative PCR was then performed for the indicated genes. Note that MYC expression is increased, while other select DDR genes demonstrated significant downregulation. *p<0.05, **p<0.01, ***p<0.001 (Student’s t tests). D. HER2+ breast cancer cells were treated with THZ531 (200 nM) for the indicated time points. Cell lysates were prepared in SDS sample buffer and subjected to fluorescent immunoblotting. E. HCC1954 cells were treated with increasing concentrations of THZ531 (0, 12.5, 25, 50,100, 200, 400, and 800 nM) for 4 hours. Cell lysates were prepared and analyzed as in (G). F. HCC1954 cells were treated with Actinomycin D at the indicated doses in the presence of vehicle control or THZ531 (400 nM). Four hours post-treatment, cells were lysed with 1x SDS sample buffer, and lysates were prepared for fluorescent immunoblotting. Merged images are shown for blots using antibodies raised in different species. The very left lane was loaded with protein markers that emit near-infrared fluorescence. G. THZ531-induced MYC expression relies on de novo protein synthesis. HCC1954 cells were treated with cycloheximide (6.25 μg/ml) and THZ531 (200 nM), either individually or in combination. Cells were harvested in 3 hours for immunoblotting. H. Tumors with low expression of CDK12 exhibit activation of a MYC signature. (Right) The consolidated MYC signature was developed by combining all 12 gene sets from MSigDB ( http://www.gsea-msigdb.org/ ) and identifying genes whose expression positively correlates with MYC across the 1305 cancer cell lines in the Cancer Cell Line Encyclopedia (CCLE, Pearson correlation r > 0.2). (Left) Expression of the 235 genes was then evaluated in CDK12, CDK7, CDK9-low ovarian tumors in comparison to their high expression counterparts. The top dot plot displays the differential expression of all 235 genes, while the bottom plot shows genes with significant differential expression (q < 0.1; n = 106, 60, 6 for the CDK12, CDK7, CDK9 groups, respectively). *p<0.05, ****p<0.0001 (Mann-Whitney test).

Article Snippet: The following primary antibodies were purchased and used for fluorescence immunoblotting: RNA polymerase II subunit B1 (phospho CTD Ser-2) Antibody, clone 3E10 (EMD Millipore, #04-1571); Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (Cell Signaling Technology, #13499); Phospho RNA Polymerase II (S2) Antibody, (Bethyl Laboratories, A300-654A); RNA polymerase II subunit B1 (phospho-CTD Ser-5) Antibody, clone 3E8 (EMD Millipore, #04-1572); Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (Cell Signaling Technology, #13523) RNA polymerase II subunit B1 (phospho-CTD Ser-7) Antibody, clone 4E12 (EMD Millipore, #04-1570); RNA Polymerase II Antibody (Bethyl Laboratories, A300-653A); RNA Polymerase II RPB1, clone 8WG16 (BioLegend, #664906); Rpb1 NTD (D8L4Y) Rabbit mAb (Cell Signaling Technology, #14958); c-Myc (clone Y69) Rabbit mAb (Abcam, #ab32072); c-Myc (D84C12) Rabbit mAb (Cell Signaling Technology, #5605); anti-CDK12 (Cell Signaling Technology, #11973); anti-CDK12 (proteintech, #26816-1-AP); anti-CDK12, clone 45F7-H2 (BIO-RAD, #VMA00874); anti-CDK13, clone 46B7-G7 (BIO-RAD, #VMA00875); CDK7 Recombinant Monoclonal Antibody (BL-80-5D4) (Bethyl, #A700-006); CDK7 (MO1) Mouse mAb (Cell Signaling Technology, #2916); CDK8 (D6M3J) Rabbit mAb (Cell Signaling Technology, #17395); CDK9 (C12F7) Rabbit mAb (Cell Signaling Technology, #2316); Anti-CDK19 (Sigma-Aldrich, #HPA007053); JunB Rabbit mAb, clone ARC0268 (ABclonal, #A4848); c-Jun (60A8) Rabbit mAb (Cell Signaling Technology, #9165); c-Fos (9F6) Rabbit mAb (Cell Signaling Technology, #2250); Anti-c-ErbB2/c-Neu (Ab-3) Mouse mAb (3B5) (EMD Millipore, #OP15); anti-PARP (Cell Signaling Technology, #9542); anti-β-Actin, clone AC-15 (Sigma, A5441); anti-Vinculin (Sigma, V9131); anti-β-Tubulin (BioLegend, # 903401).

Techniques: RNA Sequencing, Inhibition, RNA Extraction, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control, Fluorescence, Activation Assay, Comparison, Quantitative Proteomics, MANN-WHITNEY