Journal: EMBO Molecular Medicine

Article Title: CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia

doi: 10.15252/emmm.202114990

Figure Lengend Snippet: Gene expression of GPR56 , SMO (left), and TGFb targets SRC, ZEB1, and ZEB2 (right) normalized to GAPDH in AML‐491 cells determined by q‐RT‐PCR 4 h after treatment with THZ1 0.5 µM or LDC4297 2 µM. Unpaired t ‐test. Mean, SD, and individual values from three individual treatments. *** P < 0.0005, ** P < 0.005, * P < 0.05. Setup of in vivo drug treatment. NSG mice were injected with 10 5 AML 04H112 cells. Four weeks post injection, bone marrow (BM) was analyzed for human leukemic engraftment by BM aspiration. Treatment with either THZ1 or vehicle was started in the following week as indicated. BM was analyzed again after the end of the 4‐week treatment period. Overall percentage of human (huCD45 + ) leukemic cells in mice before and at the end of the 4‐week treatment period. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005. N = 10 mice for each group. Left : Representative FACS plots showing the typical CD34 low GPR56 high profile of sample 04H112 before injection and after the 4‐week treatment with THZ1. Right : both LSC compartments, the CD34 ‐ GPR56 + and the CD34 + GPR56 + fractions were significantly reduced in vivo in the THZ1 treatment group. Individual mice and mean engraftment are shown. Unpaired t ‐test. *** P < 0.0005, ** P < 0.005, * P < 0.05. N = 10 mice for each group. Left : Overall percentage of human (huCD45 + ) leukemic cells and the fraction of CD34 + GPR56 + among human cells (mean, individual mice) two weeks after drug withdrawal. Right : FACS plot visualizing that the CD34 + GPR56 + is re‐established upon drug removal. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005, * P < 0.05. Correlation plot showing an anti‐correlation between the percentage of CD34 + GPR56 + cells in AML samples and the corresponding IC50s for THZ1 determined in the respective samples. Pearson correlation. Each dot represents one sample. See Dataset for sample characteristics. FACS histogram plot (left) and summary bar graph from three replicate wells (right) showing reduction of GPR56 surface expression on the GPR56‐positive AML cell line HEL only in conditions that contain the more specific CDK7 inhibitor YKL‐5‐124, but not those that contain only the specific CDK12/13 inhibitor THZ531. Unpaired t ‐test, bars, and error bars represent mean and SD of three individual treatments, *** P < 0.0005, ** P < 0.005, * P < 0.05. SRF reporter assay showing dose‐dependent suppression of the GPR56‐CTF‐induced SRF signal by the CDK7 inhibitor YKL‐5‐124, but not by the CDK12/13 inhibitor THZ531. Four technical replicates. One of the four individual experiments. Unpaired t ‐test, bars and error bars represent mean and SD, *** P < 0.0005, ** P < 0.005, * P < 0.05.

Article Snippet: For in vivo THZ1 treatment, THZ1(#HY‐80013) was purchased from MedChemExpress and dissolved in DMSO at 300 mg/ml stock concentration.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vivo, Injection, Reporter Assay

Journal: EMBO Molecular Medicine

Article Title: CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia

doi: 10.15252/emmm.202114990

Figure Lengend Snippet: Left: Eleven primary AML specimens with different cytogenetic and molecular genetic aberrations (see Dataset for details) were treated with THZ1 or YKL‐5‐124 alone or in combination with venetoclax. IC50 values in presence of increasing doses of venetoclax are visualized for THZ1. The gray‐scale bar indicates the fraction of viable cells in presence of a high dose of venetoclax (500 nM) compared with DMSO to provide an approximation whether a sample was rather sensitive or resistant against venetoclax alone. Note that the addition of venetoclax reduces the IC50 in all samples regardless of their baseline resistance against venetoclax alone. The last four columns provide the average and maximum BLISS scores reached by a combination of THZ1 and venetoclax ( N = 11) Middle : representative dose‐response curves of THZ1 in presence of increasing doses of venetoclax for one primary AML specimen with intermediate venetoclax sensitivity. Right: representative 3D‐synergy plot showing the BLISS synergy scores at indicated concentrations of THZ1 and venetoclax for AML E204098. Dose‐response curve for the CDK7i CT7001 in presence of increasing doses of venetoclax for primary AML E2113590. The indicated doses on the right represent the IC50 dose. Schematic visualizing the setup for the combinatory in vivo treatment. AML‐661 cells were injected in NSG mice. Bones indicate the timepoints of bone marrow aspirations to monitor engraftment of human leukemic cells in mice before treatment start and during treatment. Percentage of human CD45 + leukemic cells three weeks post injection, i.e. before treatment start (left), and 4 and 6 weeks after treatment start (right). Dots represent individual mice, horizontal lines represent means. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005. Percentage of human CD34 + GPR56 + cells in the mouse bone marrow at 4 and 6 weeks post treatment start with the indicated compounds. Dots represent individual mice, horizontal lines represent means. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005, * P < 0.05. Left : Representative FACS plots showing CD34 and GPR56 expression on AML cells after 4‐week treatment with the indicated compounds. Right : Statistical analysis of the geometric mean intensity of CD34 APC (left) and GPR56 PE (right) in the four treatment groups. Horizontal lines represent means. Unpaired t ‐test, *** P < 0.0005, ** P < 0.005, * P < 0.05. Cartoon visualizing how CDK7i and venetoclax synergize to suppress both GPR56 + compartments in AML.

Article Snippet: For in vivo THZ1 treatment, THZ1(#HY‐80013) was purchased from MedChemExpress and dissolved in DMSO at 300 mg/ml stock concentration.

Techniques: In Vivo, Injection, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists

doi: 10.1073/pnas.1815780116

Figure Lengend Snippet: THZ1 inhibits Gli transcription and growth of Ptch1-deficient mouse medulloblastoma; (A) qRT-PCR of Gli1 and Gli2 in four Ptch1-deficient mouse MB lines treated as indicated for 8 h. Data are shown as mean ±SD. (B) Immunoblot detecting Gli1 expression in cell extracts from mouse MB cells treated with 0.1 μM THZ1 for 24 h. Anti–β-tubulin immunoblots are shown as loading control. (C) Dose–response curves of mouse MB lines and control cells in response to THZ1. Cells were plated in 96-well plate in triplicate and treated with THZ1 at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD. (D) Mouse MB lines were treated with THZ1 at indicated concentrations. Cell viabilities were measured at day-0/1/2/3 posttreatment and normalized to day-0 value. Data are shown as mean ±SD. (E and F) Cell proliferation and apoptosis analyses of mouse MB lines treated as indicated by EdU incorporation or Annexin-V staining FACS assay. Percentages of EdU+ (E) or Annexin-V+ (F) cells are presented.

Article Snippet: Additionally, THZ1-R (HY-19988), JQ1 (HY-13030), Ko-143 (HY-10010), and CT7001 (HY-103712A) were purchased from MedChem Express.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Control, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists

doi: 10.1073/pnas.1815780116

Figure Lengend Snippet: THZ1 targets CDK7 to inhibit Gli transcription and growth of the Hh-driven mouse medulloblastoma. (A) Immunoblot-detecting phosphorylation levels at Ser2, Ser5, and Ser7 of RNAPII CTD as well as total RNAPII from GNP, Sufu−/− MEF, SMB21, or SMB56 cells treated with THZ1 at indicated concentrations for 8 h. Anti–β-tubulin immunoblot are shown as loading control. (B) qRT-PCR of Gli1, Gli2 in SMB21 (Top) or SMB56 cells (Bottom) stably expressing Cas9 together with sgCdk7-1, sgCdk7-2, sgLacZ, or sgGFP. Data are shown as mean ±SD. (C) Immunoblot detecting Gli1 and Cdk7 in cell extracts from SMB21 (Top) or SMB56 cells (Bottom) stably expressing Cas9 together with sgCdk7-1, sgCdk7-2, sgLacZ, or sgGFP. Anti–β-tubulin immunoblot are shown as loading control. (D) In vitro growth of SMB21 (Top) or SMB56 cells (Bottom) stably expressing Cas9 together with sgCdk7-1, sgCdk7-2, sgLacZ, or sgGFP were measured at day-0/2/4 and normalized to day-0 value. Data are shown as mean ±SD.

Article Snippet: Additionally, THZ1-R (HY-19988), JQ1 (HY-13030), Ko-143 (HY-10010), and CT7001 (HY-103712A) were purchased from MedChem Express.

Techniques: Western Blot, Control, Quantitative RT-PCR, Stable Transfection, Expressing, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists

doi: 10.1073/pnas.1815780116

Figure Lengend Snippet: THZ1 suppresses Gli transcription and growth of Hh-driven mouse medulloblastoma with SMOi resistance. (A) qRT-PCR of Gli1, Gli2 in SmoWT-MB or SmoD477G-MB cells treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 8 h. Data are shown as mean ±SD. (B) SmoWT-MB and SmoD477G-MB cells were treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD. (C) qRT-PCR of Gli1, Gli2 in SMB21-Mock, SMB21 cells stably expressing shCtrl (SMB21-shCtrl), or shSufu (SMB21-shSufu) treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 8 h. Data are shown as mean ±SD. (D) SMB21-Mock, SMB21-shCtrl, and SMB21-shSufu cells were treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD. (E) qRT-PCR of Gli1, Gli2 in SMB21-Mock, SMB21 cells stably expressing empty vector (SMB21-EV) or GLI2-deltaN (SMB21-GLI2-deltaN) treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 8 h. Data are shown as mean ±SD. (F) SMB21-Mock, SMB21-EV, and SMB21-GLI2-deltaN cells were treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD.

Article Snippet: Additionally, THZ1-R (HY-19988), JQ1 (HY-13030), Ko-143 (HY-10010), and CT7001 (HY-103712A) were purchased from MedChem Express.

Techniques: Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists

doi: 10.1073/pnas.1815780116

Figure Lengend Snippet: THZ1 and JQ1 works synergistically together to suppress Gli transcription and growth of Hh-driven mouse medulloblastoma. (A–D) Cell viability and synergy measurement in combinatory drug treatments. (Top) SmoWT-MB (A), SmoD477G-MB (B), SMB21 (C), SMB21-shSufu (D) cells were treated with THZ1 and JQ1 individually or in combination at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD. (Bottom) CI value of each drug combination condition was calculated using CalcuSyn software and a CI value of less than 1 indicates synergy. qRT-PCR of Gli1, Gli2 in SMB21 (E) or SMB21-shSufu (F) cells treated with THZ1 and JQ1 individually or in combination at indicated concentrations for 8 h. Data are shown as mean ±SD.

Article Snippet: Additionally, THZ1-R (HY-19988), JQ1 (HY-13030), Ko-143 (HY-10010), and CT7001 (HY-103712A) were purchased from MedChem Express.

Techniques: Software, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists

doi: 10.1073/pnas.1815780116

Figure Lengend Snippet: CDK7 inhibition effectively suppresses Gli transcription and growth of SMOi-responsive or -resistant Hh-driven mouse medulloblastoma in vivo. Tumor growth of allografts established by injecting SMB21 (A) or SMB56 cells (B) stably expressing Cas9 together with sgCdk7-1, sgCdk7-2, sgLacZ, or sgGFP as indicated into hind flanks of nude mice (five mice with 10 allografted tumors in each group) was assessed by caliper measurement. Data are shown as mean ±SEM. P values were determined by two-way ANOVA. Nude mice with SMB21 (C) or SMB21-shSufu (D) flank allografts (each mouse carried two allografts) were treated with THZ1 at 10 mg/kg or DMSO twice daily by i.v. injection. Tumor growth assessed by caliper measurement is presented as mean ±SEM. P value was determined by two-way ANOVA. (E) Nude mice with SMB21-shSufu intracranial allografts were treated with THZ1 at 10 mg/kg (n = 8) or DMSO (n = 6) twice daily by i.v. injection as indicated. Mice survival was monitored and Kaplan–Meier survival curve was presented. P value was determined by log-rank test (Mantel–Cox test). (F) Nude mice with SMB21-shSufu flank allografts (each mouse carried two allografts) were treated with THZ1 (10 mg/kg, twice a day, i.v.) and JQ1 (25 mg/kg, twice a day, i.p.) individually or in combination as indicated. Tumor growth assessed by caliper measurement is presented as mean ±SEM. P values were determined by two-way ANOVA. qRT-PCR of Gli1, Gli2 in SMB21 (G) or SMB21-shSufu (H) allografts treated with THZ1 or DMSO (n = 4). Data are shown as mean ±SEM. (I) qRT-PCR of Gli1, Gli2 in SMB21-shSufu flank allografts treated with THZ1 and JQ1 individually or in combination (n = 4). Data are shown as mean ±SEM.

Article Snippet: Additionally, THZ1-R (HY-19988), JQ1 (HY-13030), Ko-143 (HY-10010), and CT7001 (HY-103712A) were purchased from MedChem Express.

Techniques: Inhibition, In Vivo, Stable Transfection, Expressing, Injection, Quantitative RT-PCR

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists

doi: 10.1073/pnas.1815780116

Figure Lengend Snippet: THZ1 suppresses GLI transcription and growth of Hh-driven human tumors with SMOi resistance. A673 (A) or ATRT-03 (B) cells were treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD. qRT-PCR of GLI1 in A673 (C) or ATRT-03 (D) cells treated with THZ1, JQ1, or GDC-0449 at indicated concentrations for 8 h. Data are shown as mean ±SD. A673 (E) and ATRT-03 (F) cells were treated with THZ1 and JQ1 individually or in combination at indicated concentrations for 72 h before cell viabilities were assessed. Data are shown as mean ±SD on the top. CI value of each drug combination condition was calculated using CalcuSyn software and presented on the bottom. A CI value of less than 1 indicates synergy. qRT-PCR of GLI1 in A673 (G) and ATRT-03 (H) cells treated with THZ1 and JQ1 individually or in combination at indicated concentrations for 8 h. Data are shown as mean ±SD.

Article Snippet: Additionally, THZ1-R (HY-19988), JQ1 (HY-13030), Ko-143 (HY-10010), and CT7001 (HY-103712A) were purchased from MedChem Express.

Techniques: Quantitative RT-PCR, Software

Journal: eLife

Article Title: The TFIIH complex is required to establish and maintain mitotic chromosome structure

doi: 10.7554/eLife.75475

Figure Lengend Snippet: ( A ) Western blot for CAP-E, XPB, and Tubulin in Mock or CAP-E depleted extracts. Dilution series of Mock-depleted extracts was used to calculate the % depletion of CAP-E using quantitative fluorescence. ( B ) 1% agarose gel stained with SYBR safe stain showing RNase A dependent removal of total RNAs from high-speed supernatant egg extracts. ( C ) Representative fluorescence images of chromatid assembly at steady state (180 min after sperm addition) in the presence of indicated buffers and enzymes. See Materials and methods for details. Note that the high salt in the RNase H buffer causes morphological changes to chromatids regardless of activity. Mean condensation parameters for each condition are indicated in lower left corner of each image. ( D ) Western blot for XPB and Tubulin in IgG or XPB-depleted extracts. Dilution series of IgG-depleted extracts was used to calculate the % depletion of XPB using quantitative fluorescence. ( E ) Western blot for XPF, ERCC1, and Tubulin in IgG or ERCC1-depleted extracts. Dilution series of IgG-depleted extracts was used to calculate the % depletion of ERCC1 and XPF using quantitative fluorescence. ( F ) Representative fluorescence images of chromatid assembly at steady state (180 min after sperm addition) in the presence of DMSO, 30 μM CDK7 inhibitor THZ1, or 30 μM THZ1 and 50 μM triptolide (TPL). ( G ) Triptolide does not affect CDK1 activation or maintenance of M phase. Western blot for CDK1 T161ph, histone H3T3ph, and Tubulin in M phase HSS extracts in the presence of DMSO or 50 μM triptolide. Both CDK1T161ph (a CDK7 substrate) and H3T3ph are only highly phosphorylated in M phase, and their appearance is not affected by triptolide treatment. Figure 1—figure supplement 1—source data 1. Source data for .

Article Snippet: Chemical compound, drug , THZ1 , Cayman , Cat# 9002215–1 , 30 μM.

Techniques: Western Blot, Fluorescence, Agarose Gel Electrophoresis, Staining, Activity Assay, Activation Assay

Journal: eLife

Article Title: The TFIIH complex is required to establish and maintain mitotic chromosome structure

doi: 10.7554/eLife.75475

Figure Lengend Snippet: ( A ) Representative immunofluorescence images of Xenopus sperm nuclei incubated in HSS extracts for 180 min. DMSO was added at 25 min as a control. Chromatids were labeled with Hoechst, either anti-Pol II S5ph (paused) or anti-Pol II S2ph (elongating), and co-stained with anti-CENP-A antibodies. ( B ) Representative immunofluorescence images of Xenopus sperm nuclei incubated in DMSO, triptolide (TPL), THZ1, DRB, or alpha-amanitin treated HSS extracts for 180 min. All inhibitors were added 25 min after sperm addition. Chromatids were labeled with Hoechst, anti-Pol II S5ph, or anti-Pol II S2ph antibodies.

Article Snippet: Chemical compound, drug , THZ1 , Cayman , Cat# 9002215–1 , 30 μM.

Techniques: Immunofluorescence, Incubation, Control, Labeling, Staining

Journal: eLife

Article Title: The TFIIH complex is required to establish and maintain mitotic chromosome structure

doi: 10.7554/eLife.75475

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , THZ1 , Cayman , Cat# 9002215–1 , 30 μM.

Techniques: Immunodepletion, Control, Recombinant, Immunoprecipitation, Staining, Western Blot, Software

Journal: bioRxiv

Article Title: Elongation factor ELOF1 drives transcription-coupled repair and prevents genome instability

doi: 10.1101/2021.05.11.443558

Figure Lengend Snippet: (A) Left panel: Schematic of the genomic locus of CSB and used strategy for generating the homozygous CSB-mScarletI-HA KI cell line. Half arrows indicate primer locations. Middle and right panel: Genotyping PCR and immunoblot for CSB -KI cell line. (B) Left panel: Schematic of the genomic locus of UVSSA and used strategy for generating the homozygous UVSSA-mScarletI-HA KI cell line. Half arrows indicate primer locations. Middle and right panel: Genotyping PCR and immunoblot for UVSSA -KI cell line. (C) Left panel: CSB mobility was determined by FRAP analysis of CSB-mScarletI after the indicated treatments. THZ1: 1 hour treatment (2 µM) before UV-C irradiation (4 J/m 2 ) or mock treatment. Right panel: Relative immobile fraction of CSB as determined by FRAP analysis. Plotted values represent mean ± SEM and are normalized to mock treated. n≥15 cells. (D) Same as C but for UVSSA-mScarletI. n≥10 cells. (E+F) FRAP analyses of CSB-mScarletI (E) or UVSSA-mScarletI (F) mobility after transfection with indicated siRNAs in individual graphs. Cells were mock treated (NT) or analyzed directly (UV) or 5 hours (5hr UV) after irradiation with 4 J/m 2 UV-C. (G) Relative fluorescence intensity of UVSSA in UVSSA -KI cells transfected with indicated siRNAs as determined by live-cell imaging. Plotted values represent mean ± SEM. n≥16 cells. (H) FRAP analysis of CSB in CSB -KI cells transfected with indicated siRNAs 2 hours after UV. VCPi: VCP inhibitor (5 µM) was directly added after UV-C (4 J/m 2 ). (I) Immunoblot of chromatin fraction of indicated cell lines 1 hour after 12 J/m 2 UV-C or mock treatment. NAEi = 1 hour treatment with NEDDylation inhibitor (10 µM). SSRP1 is shown as loading control. (J) Immunoblot of chromatin fraction of HCT116 cells transfected with indicated siRNAs 1 hour after 12 J/m 2 UV-C or mock treatment. SSRP1 is shown as loading control.

Article Snippet: Cell were treated for 1 hour with the following chemicals: Actinomycin D (Sigma, 1 µg/ml), Flavopiridol (Sigma, 1 µM), THZ1 (Xcessbio, 2 µM), Mitomycin C (Sigma, 10 µg/ml unless indicated differently), or potassium bromate (KBrO 3 , Sigma).

Techniques: Western Blot, Irradiation, Transfection, Fluorescence, Live Cell Imaging

Journal: Genes & Development

Article Title: CDK7 regulates organ size and tumor growth by safeguarding the Hippo pathway effector Yki/Yap/Taz in the nucleus

doi: 10.1101/gad.333146.119

Figure Lengend Snippet: CDK7 regulates organ size and tumor progression through Yap/Taz. ( A ) Western blot analysis of Yap and CDK7 in MDA-MB-231 cells transfected with siControl or siCDK7 and infected with lentiviruses expressing the empty vector, wild-type Yap, or the S128D mutant. ( B ) Relative mRNA levels of the indicated Hippo target genes in the indicated MDA-MB-231 cells were measured by RT-qPCR. Data are means ± SD. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001 (Student's t -test). ( C ) Relative cell numbers of MDA-MB-231 cells treated with siControl or siCDK7 and infected with lentiviruses expressing wild-type or S128D Yap were measured by the WST-1 assay at different time points after infection. Data are means ± SD. (**) P < 0.01; (***) P < 0.001 (Student's t -test). ( D , E ) Transwell invasion assay of MDAMB-231 cells treated with siControl or siCDK7 and infected with lentiviruses expressing wild-type or S128D Yap. Data are means ± SD. (**) P < 0.01; (***) P < 0.001 (Student's t -test). ( F ) Western blot analysis of Yap in MDA-MB-231 cells treated with THZ1. ( G ) Relative mRNA levels of the indicated Hippo target genes in MDA-MB-231 cells treated with THZ1. Data are means ± SD. (***) P < 0.001 (Student's t -test). ( H ) Tumor size of the indicated MDA-MB-231 xenografts in mice treated with vehicle or THZ1 at the indicated time after drug treatment. ( I ) Macroscopic images of tumors derived from the indicated MDA-MB-231 xenografts at the end of drug treatment. ( J ) Quantification of tumor weight of the indicated MDA-MB-231 xenografts at the end of drug treatment. Data are means ± SD. n = 7 mice for each group. (NS) not significant; (**) P < 0.01; (***) P < 0.001 (Student's t -test). ( K ) MST1/2 DKO mice were treated with vehicle or THZ1 (10 mg/kg) every other day starting at the age of 1 mo until 3 mo old. Representative macroscopic images of livers were shown. ( L ) Quantitative analysis of liver-to-body weight ratio. Data are means ± SD. n = 6 mice for each group. (***) P < 0.001 (Student's t -test). ( M ) Western blot analysis of control and THZ1-treated liver tissues of the indicated genotypes using the indicated antibodies. ( N , O , Q ) Liver cross-sections of the indicated genotypes treated with vehicle or THZ1 were subjected to immunostaining with anti-EPCAM, SOX9, F4/80, or Ki67 antibody. Scale bar, 100 μm. ( P ) Quantification of Ki67 + cells in liver sections of the indicated genotypes treated with vehicle or THZ1. Data are means ± SD. n = 5 sections for each group. (***) P < 0.001 (Student's t -test).

Article Snippet: Mice were treated with THZ1 (APEXBIO, catalog no. A8882) by intraperitoneal injection at a concentration of 10 mg/kg every other day or in equivalent volumes of vehicle as a control.

Techniques: Western Blot, Transfection, Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, WST-1 Assay, Transwell Invasion Assay, Derivative Assay, Control, Immunostaining

Journal: Genes & Development

Article Title: CDK7 regulates organ size and tumor growth by safeguarding the Hippo pathway effector Yki/Yap/Taz in the nucleus

doi: 10.1101/gad.333146.119

Figure Lengend Snippet: Targeting CDK7 for treating Yap/Taz-driven cancer. ( A ) A conserved role of CDK7/CRL4 DCAF12 in the regulation of nuclear Yki/Yap/Taz. CDK7 phosphorylates Yki/Yap/Taz to prevent its binding to CRL4 DCAF12 , thereby stabilizing Yki/Yap/Taz in the nucleus. ( B , top ) Yap/Taz/TEAD binds superenhancers together with other transcriptional factors (X, Y, etc.) to drive the expression of genes essential for tumor growth. ( Bottom ) CDK7 inhibitor THZ1 blocks tumor growth by inhibiting both Yap/Taz and Pol II in Yap/Taz-driven cancer.

Article Snippet: Mice were treated with THZ1 (APEXBIO, catalog no. A8882) by intraperitoneal injection at a concentration of 10 mg/kg every other day or in equivalent volumes of vehicle as a control.

Techniques: Binding Assay, Expressing

Journal: eLife

Article Title: Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations

doi: 10.7554/eLife.59770

Figure Lengend Snippet: ( A ) Treatment of HeLa cells with small-molecule inhibitors reveals that alpha-satellite transcripts are mediated by RNA polymerase II. Cells were treated with the RNA Polymerase I inhibitor BMH-21 (24 hr), the RNA Polymerase III inhibitor ML-60218 (24 hr), or the Cdk7 inhibitor THZ1 (5 hr), which inhibits RNA Polymerase II initiation. Transcripts were identified using the ASAT smFISH probe set. ( B ) Quantification of smFISH foci from ( A ) after treatment of HeLa cells with small-molecule inhibitors against Cdk7, RNA Pol I, and RNA Pol III. smFISH foci were substantially reduced after inhibition of RNA Pol II activator, Cdk7, but increased by RNA Pol I inhibition. Error bars represent the mean and standard deviation of at least 240 cells. ( C ) Graph showing independent replicates of ASAT smFISH foci for each small-molecule inhibitor treatment (Cdk7, RNA Pol I, and RNA Pol III). P-values represent T-tests for the indicated comparisons. ( D ) RT-qPCR quantification reveals significantly reduced levels of chromosome 21 alpha-satellite transcripts of cells treated by the Cdk7 inhibitor THZ1 for 5 hr, but increased levels following RNA polymerase I inhibition (24 hr treatment) when compared to control HeLa cells. The levels of alpha-satellite RNA from chromosome 21 detected was outside of our quantifiable range in cells treated with CDK7 inhibitor and thus was set to 0. The mean of 3 biological replicates was plotted and error bars represent the standard deviation. P-value represents the results of a T-test. Figure 3—source data 1. Source data for the RT-qPCR experiments shown in .

Article Snippet: For inhibitor experiments, RNA Polymerase inhibitors were added to cells at the following concentrations: BMH-21 (RNAPI inhibitor; Millipore) at 1 μM; ML-60218 (RNAP III inhibitor; Fisher) at 20 μM; THZ1 (Cdk7 Inhibitor; Fisher Scientific) at 1 μM.

Techniques: Inhibition, Standard Deviation, Quantitative RT-PCR, Control

Journal: eLife

Article Title: Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations

doi: 10.7554/eLife.59770

Figure Lengend Snippet: ( A ) Immunofluorescence images (using anti-CENP-C, anti-CENP-A and anti-CENP-B antibodies) confirming the effective elimination of the corresponding gene targets throughout the population. Control cells (top) and inducible knockout cells (bottom). Scale bar, 20 µm. ( B ) Analysis of alpha-satellite smFISH transcripts (ASAT probe set) for cells depleted for diverse cell division components; proteins involved in centromere regulation (Sgo1 and BubR1), DNA replication (Mcm6, Gins1, Orc1, and Cdt1), sister chromatid cohesion (ESCO2, Scc1), chromosome condensation (Smc2, CAPG, CAPG2, TOP2A), and nucleosome remodeling (SSRP1) were targeted using Cas9. Despite the diverse roles of these proteins in different aspects of centromere function, none of these inducible knockouts resulted in reduced levels of ASAT alpha-satellite transcripts as detected by smFISH analysis. Error bars represent the mean and standard deviation of at least 240 cells. ( C ) The increase in alpha-satellite transcripts in cells depleted for CENP-C depends on RNA Polymerase II as THZ1 treatment resulted in a substantial reduction in smFISH foci in both control cells and CENP-C inducible knockout cells using SF1 probes.

Article Snippet: For inhibitor experiments, RNA Polymerase inhibitors were added to cells at the following concentrations: BMH-21 (RNAPI inhibitor; Millipore) at 1 μM; ML-60218 (RNAP III inhibitor; Fisher) at 20 μM; THZ1 (Cdk7 Inhibitor; Fisher Scientific) at 1 μM.

Techniques: Immunofluorescence, Control, Knock-Out, Standard Deviation

Journal: eLife

Article Title: Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations

doi: 10.7554/eLife.59770

Figure Lengend Snippet: ( A ) Quantification of smFISH foci (ASAT probe set) after elimination of selected centromere and kinetochore components reveals that centromere components are not required for the production of alpha-satellite transcripts. Inducible knockouts were generated using Cas9 using previously described cell lines ( ; ). Notably, inducible knockout of CENP-C results in a substantial increase in smFISH foci. Error bars represent the mean and standard deviation of at least 240 cells. ( B ) Representative images showing the substantial increase in smFISH foci after elimination of the centromere component CENP-C. ( C ) Quantification of ASAT smFISH foci under the indicated conditions. The increase in alpha-satellite transcripts in cells depleted for CENP-C depends on RNA Polymerase II, as THZ1 treatment (Cdk7 inhibition; 5 hr) resulted in a substantial reduction in smFISH foci in both control cells and CENP-C inducible knockout cells. ( D ) Quantification of smFISH foci in CENP-C inducible knockout RPE-1 cells reveals that the increase in alpha-satellite transcripts following CENP-C knockout is not specific to HeLa cells. Error bars represent the mean and standard deviation of at least 170 cells. ( E ) RT-qPCR for alpha-satellite transcripts from chromosome 21 indicates a substantial increase in steady state alpha-satellite RNA levels in HeLa CENP-C inducible knockout cells. The mean of three biological replicates for control and four biological replicates for the CENP-C inducible knockouts was plotted. Error bars represent the standard deviation. P-value represents the results of a T-test. ( F ) Quantification of smFISH foci number in CENP-C inducible KO cells and Pol I-inhibited (24 hr treatment) cells compared to HeLa cell controls. ( G ) Quantification of the intensity of individual smFISH foci from the same experiment tested in F showing similar intensities despite the increase in foci number. ( H ) The half-life of alpha-satellite RNAs derived from chromosome 21 was determined in HeLa and CENP-C inducible knockout cells by RT-qPCR various times following RNA polymerase II inhibition (THZ1 treatment). The level of chromosome 21 alpha-satellite RNA was normalized to GAPDH, a stable mRNA. The half-life of these centromeric transcripts is 78 and 72 min in HeLa and CENP-C inducible knockout cells, respectively. Graph shows mean and standard deviation for two biological replicates. Scale bars, 25 µm. Figure 4—source data 1. Source data for the RT-qPCR experiments shown in .

Article Snippet: For inhibitor experiments, RNA Polymerase inhibitors were added to cells at the following concentrations: BMH-21 (RNAPI inhibitor; Millipore) at 1 μM; ML-60218 (RNAP III inhibitor; Fisher) at 20 μM; THZ1 (Cdk7 Inhibitor; Fisher Scientific) at 1 μM.

Techniques: Generated, Knock-Out, Standard Deviation, Inhibition, Control, Quantitative RT-PCR, Derivative Assay

Journal: eLife

Article Title: Alpha-satellite RNA transcripts are repressed by centromere–nucleolus associations

doi: 10.7554/eLife.59770

Figure Lengend Snippet:

Article Snippet: For inhibitor experiments, RNA Polymerase inhibitors were added to cells at the following concentrations: BMH-21 (RNAPI inhibitor; Millipore) at 1 μM; ML-60218 (RNAP III inhibitor; Fisher) at 20 μM; THZ1 (Cdk7 Inhibitor; Fisher Scientific) at 1 μM.

Techniques: Generated, cDNA Synthesis, SYBR Green Assay