Journal: Biomedicines

Article Title: Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer

doi: 10.3390/biomedicines11030928

Figure Lengend Snippet: CDK9 expression in normal and colorectal cancer tissue and cell lines. ( a ) Representative pictures of CDK9-Immunohistochemistry and HE staining in human normal and CRC tissue. A,E: CDK-9 IHC staining in normal (A) and tumor tissue (E) of patient 165; B,F: CDK-9 IHC staining in normal (B) and tumor tissue (F) of patient 89; C,G: HE staining in normal (C) and tumor tissue (G) of patient 165; D,H: HE staining in normal (D) and tumor tissue (H) of patient 89 ( b ) Statistical significance between groups was determined using Wilcoxon signed-rank test for group comparison demonstrated by a boxplot. p values of <0.05 were considered statistically significant. *** p < 0.001. ( c ) CDK9 expression was also shown in different colorectal cancer cell lines. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown.

Article Snippet: Microwave setup was 450 W for 15 min followed by 270 W for 10 min. CDK9 (C12F7) antibody was diluted 1:150 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Comparison, Western Blot

Journal: Biomedicines

Article Title: Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer

doi: 10.3390/biomedicines11030928

Figure Lengend Snippet: Correlation of CDK9 expression with clinicopathologic parameters of CRC.

Article Snippet: Microwave setup was 450 W for 15 min followed by 270 W for 10 min. CDK9 (C12F7) antibody was diluted 1:150 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing

Journal: Biomedicines

Article Title: Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer

doi: 10.3390/biomedicines11030928

Figure Lengend Snippet: Impact of CDK9 expression on survival of colorectal cancer patients ( a ) and ( b ) Kaplan-Meier survival plot displaying the overall survival of the whole cohort and T3 + T4 according to CDK9 expression in colorectal cancer tissue. Patients with cancer expressing high CDK9 levels show increased survival rates ( p = 0.087 and p = 0.044) compared to those with cancer expressing low CDK9 levels. ( c ) Representative pictures for low CDK9 expressing subgroup (HE and CDK9-immunohistochemistry), A,B: HE and CDK9 expression in patient 8, C,D: HE and CDK9 expression in patient 20; E,F: HE and CDK9 expression in patient 44. ( d ) Representative pictures for high CDK9 expressing subgroup (HE and CDK9-immunohistochemistry), A,B: HE and CDK9 expression in patient 142, C,D: HE and CDK9 expression in patient 154; E,F: HE and CDK9 expression in patient 169.

Article Snippet: Microwave setup was 450 W for 15 min followed by 270 W for 10 min. CDK9 (C12F7) antibody was diluted 1:150 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Immunohistochemistry

Journal: Biomedicines

Article Title: Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer

doi: 10.3390/biomedicines11030928

Figure Lengend Snippet: Synergistic effect of Dinaciclib treatment in combination with izTRAIL. Five colon cancer cell lines (HT-29, HCT-116, SW-480, SW-620, and DLD-1) were treated in different ways. ( a ) using izTRAIL (0, 0.1, 1, 10, 100 ng/mL) and ( b ) Dinaciclib (0, 3.125, 6.25, 12.5, 25, 50, 100 nM) for 24, 48, and 72 h. ( c ) preincubated for 4 h with the CDK9 inhibitor Dinaciclib (25 nM) and subsequently treated with izTRAIL at the indicated concentrations (0, 0.1, 1, 10, 100 ng/mL). Cell viability was analyzed by MTT viability assay. Results were normalized to DMSO-treated cells. All values are presented as means ± SD of three independent experiments. ( d ) 50,000 cells/well HT-29 seeded into 6-well plates and were treated with solvent (DMSO) or Dinaciclib (25 nM) for 1 h and subsequently stimulated with izTRAIL for 24 h. Long-term survival was visualized after 7 days by crystal violet staining. ( e ) HT-29 (2000 cells/well) were seeded into 6-well plates in soft agar supplemented with solvent (DMSO), izTRAIL (1, 10 ng/mL), and Dinaciclib (5 nM), respectively, alone or in combination (T1: 1 ng/mL izTRAIL, T10: 10 ng/mL izTRAIL, Combi 1: 1 ng/mL izTRAIL + 5 nM Dinaciclib, Combi 10: 10 ng/mL izTRAIL + 5 nM Dinaciclib). Formed colonies were photographed for three times in a bright field at specific positions located on each fan-shaped center of every well divided into three equal parts and subsequentially visualized and quantified using MTT staining after 21–28 days. One of five independent experiments is shown. ( f ) All values are presented as means ± SD. ** p < 0.01, *** p < 0.001; Student’s t -test.

Article Snippet: Microwave setup was 450 W for 15 min followed by 270 W for 10 min. CDK9 (C12F7) antibody was diluted 1:150 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: MTT Viability Assay, Solvent, Staining

Journal: Biomedicines

Article Title: Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer

doi: 10.3390/biomedicines11030928

Figure Lengend Snippet: Changes in Sub G1 phase of cell cycle and apoptosis induction in HT−29 cells under the influence of pharmacological CDK9 inhibition plus izTRAIL. ( a ) Analysis of cell cycle Sub G1 phase by propidium iodide (PI) staining for HT−29 cells. Flow cytometry was used to assess Sub G1 DNA content in the entire cell population. Untreated cells (DMSO) were used as negative control; cells were treated either with TRAIL (10 ng/mL) and Dinaciclib (25 nM) or the combination for 24 h. For each treatment condition, 10,000 events were recorded and cellular debris was excluded from the analysis. Data represent one representative of four independent experiments. ( b ) Data are presented as means ± SD; ** p < 0.01; Student’s t -test. ( c ) HT−29 cells were preincubated with DMSO or Dinaciclib (25 nM) for 12 h and subsequently stimulated with izTRAIL (10 ng/mL) for the indicated periods (0, 1, 2, 4, 8 h). Cells were lysed and subjected to western blotting. Expression levels of Caspase-8, BID, Caspase-9, Caspase-3 and PARP were determinated at the indicated timepoints in control and treated cells. β−actin was used as a loading control. One representative of three independent experiments is shown.

Article Snippet: Microwave setup was 450 W for 15 min followed by 270 W for 10 min. CDK9 (C12F7) antibody was diluted 1:150 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Inhibition, Staining, Flow Cytometry, Negative Control, Western Blot, Expressing, Control

Journal: Biomedicines

Article Title: Evaluation of CDK9 Inhibition by Dinaciclib in Combination with Apoptosis Modulating izTRAIL for the Treatment of Colorectal Cancer

doi: 10.3390/biomedicines11030928

Figure Lengend Snippet: CDK9 inhibition may overcome TRAIL resistance by suppression of c-FLIP and Mcl-1. ( a ) HT-29 cells were treated with 25 nM Dinaciclib for the indicated periods. Cells were lysed and subjected to western blotting to dermine the levels of CDK9, FADD, Caspase-8, Bid, Bax, Bak, Mcl-1, Bcl-xL, Bcl-2, Caspase-9, Caspase-3, c-FLIP, cIAP1, cIAP2, XIAP, and Survivin at the indicated timepoints. β-actin was used as a loading control. ( b ) SW-620 and DLD-1 cells were lysed and analyzed by western blotting concering the expression levels of CDK9, Mcl-1, c-FLIP. β-actin was used as a loading control. One representative of two independent experiments is shown.

Article Snippet: Microwave setup was 450 W for 15 min followed by 270 W for 10 min. CDK9 (C12F7) antibody was diluted 1:150 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Inhibition, Western Blot, Control, Expressing

Journal: Science Advances

Article Title: Targeting OXPHOS de novo purine synthesis as the nexus of FLT3 inhibitor–mediated synergistic antileukemic actions

doi: 10.1126/sciadv.abp9005

Figure Lengend Snippet: ( A to C ) Knockdown efficiency of selected targets by shRNA as detected by qPCR and Western blotting; GAPDH or β-actin serves as loading control. n = 2. * P < 0.05 and ** P < 0.01. ( D to F ) Dose-response curves of shCDK9-, shDHODH-, and shCDK7-stable MOLM-13 cells in response to 120-hour gilteritinib treatment. Cell viability was measured with MTS. Results are shown as means ± SEM of four technical replicates and three to four biological replicates. *** P < 0.0001; ns, not significant. ( G ) Cell counts of MOLM-13 cells at different time points in different treatment groups. Results are shown as means ± SEM of three biological replicates. ( H ) Knockdown of CDK9 or DHODH but not CDK7 with shRNA increases the frequency of apoptotic AML cell lines with gilteritinib. Parental, scrambled, or gene-targeting shRNA-stable MOLM-13 cells were treated with 8 nM gileritinib for 120 hours and stained with annexin V/PI for flow cytometry analysis. Data are shown as means ± SEM of % population from triplicates.

Article Snippet: Anti-CDK7 (MO1), CDK9 (C12F7), PRMT5 (D5P2T), DHODH (E9X8R), adolase A (D73H4), hexokinase I (C35C4), hexokinase II (C64G5), PFKFB3 (D7H4Q), enolase-1, enolase-2 (E2H9X), PKM1 (D30G6), PKM2 (D78A4), LDHA (C4B5), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11), PFAS, SDHA, UQCRQ, and GMPS antibodies were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Knockdown, shRNA, Western Blot, Control, Staining, Flow Cytometry

Journal: Science Advances

Article Title: Targeting OXPHOS de novo purine synthesis as the nexus of FLT3 inhibitor–mediated synergistic antileukemic actions

doi: 10.1126/sciadv.abp9005

Figure Lengend Snippet: ( A ) Synergistic effect of pairwise dose combinations of gilteritinib and dinaciclib, EPZ015666 or brequinar on MOLM-13 and MV4-11 cells. For dinaciclib, cells were treated for 2 hours with the drug before it was washed off, and all other drugs were incubated with cells for 96 hours. Cell viability was measured with MTS. HSA analysis was used to determine regions of synergy. ( B ) Brequinar and gilteritinib synergistically suppress cell proliferation. MOLM-13 and MV4-11 cells were treated with vehicle, 50 nM or 100 nM brequinar in combination with vehicle, 4 or 8 nM gilteritinib for 96 hours before cells were fixed and permeabilized for intracellular BV421–anti-Ki67 staining and flow cytometry analysis. ( C and D ) Nucleoside cocktail rescued drug combination–induced apoptosis and proliferation deficiencies of MOLM-13 and MV4-11 cells at 96 hours. Cell apoptosis and proliferation were measured with annexin V/PI staining and BV421–anti-Ki67 staining, respectively. The percentage of viable cells is expressed as means ± SEM. Results are representative of triplicates. * P < 0.05. NT, nucleoside cocktail consisting of adenosine, uridine, inosine, cytosine, guanosine, and thymidine (20 μM each). ( E ) Gilteritinib in combination with either EPZ015666, dinaciclib, or brequinar eliminate self-renewal potentials of FLT3 -ITD primary AML cells while sparing healthy hematopoietic stem cells. Results are shown as means of two biological replicates. * P < 0.05 in comparison with single agents. ( F ) Synergistic effect of gilteritinib in combination with CDK9 selective inhibitors (DIN, dinaciclib; PHA, PHA-767491; BAY, BAY-1143572) on apoptosis of FLT3 -ITD primary AML cells. Primary cells were treated with vehicle, single agents, or drug combinations for 96 hours with stroma coculture before AML cell apoptosis was measured with annexin V/PI staining. Results are shown as means ± SEM of three biological replicates.

Article Snippet: Anti-CDK7 (MO1), CDK9 (C12F7), PRMT5 (D5P2T), DHODH (E9X8R), adolase A (D73H4), hexokinase I (C35C4), hexokinase II (C64G5), PFKFB3 (D7H4Q), enolase-1, enolase-2 (E2H9X), PKM1 (D30G6), PKM2 (D78A4), LDHA (C4B5), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11), PFAS, SDHA, UQCRQ, and GMPS antibodies were purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Incubation, Staining, Flow Cytometry, Comparison

Journal: Nucleic Acids Research

Article Title: Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution

doi: 10.1093/nar/gkac404

Figure Lengend Snippet: The specific lack of SNORD13-mediated acetylation in helix 45 does not alter cell growth or translation. ( A ) Schematic representation of SNORD13 locus at human chromosome 8. The relative location of the two DNA sequences targeted by sgRNAs is shown (PAM sequence is underlined). ( B ) Northern blot showing that SNORD13 is no longer detected in clones bearing deletions. * indicates truncated SNORD13 form which is routinely detected and corresponding to RNA species whose 5′-end is positioned 5–6 nucleotides upstream of the C-box (not shown). ( C ) RP-HPLC chromatograms of nucleosides obtained from helix 45 of WT (black) and SNORD13-KO (red) cells. Peak corresponding to SSU-ac 4 C1842 is indicated. ( D ) Sanger DNA sequencing of RT-PCR products obtained after borohydride treatments of total RNA extracted from WT and SNORD13-KO cells. Percentage of misincorporation (C-to-U) at SSU-C1842 (helix 45) and SSU-C1337 (helix 34) is indicated above each electropherogram. ( E ) Growth curves of WT and SNORD13-KO cells as judged by cell counting ( n = 6 for each genotype). ( F ) Apoptosis levels determined by flow cytometry using propidium and Annexin V staining of WT and SNORD13-KO cells ( n = 2 for each genotype). ( G ) SUnSET assay. Incorporation of puromycin into the elongating peptide was assayed in WT and SNORD13-KO cells (western blotting with anti-puromycin antibodies). Ponceau S staining of the membrane and detection of CDK9 (western blotting) were used as gel loading controls. Cells treated with the translation inhibitor cycloheximide (CHX) were also used as negative controls. ( H ) Luciferase reporter gene assays. WT and SNORD13-KO cells were transiently co-transfected by two plasmids expressing Renilla luciferase gene (LucR; internal control) or Firefly luciferase gene (lucF; translation reporter) carrying either an in-frame stop-codons (UAA, UAG), a detrimental mutation (R-to-S) or an IRES from FGF1A, FGF2 and CMV. Histograms show the normalized luciferase activity (lucF-to-lucR ratio). Data are expressed as mean ± s.e.m. and represent 5–6 independent experiments with triplicate measurements.

Article Snippet: Total cell extracts were then processed for western blot using an anti-puromycin antibody (Millipore, clone 12D10 #MABE343) as well as an Anti-CDK9 (C12F7) mAb (cellsignal.com) as a gel loading control.

Techniques: Sequencing, Northern Blot, Clone Assay, DNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Cell Counting, Flow Cytometry, Staining, Western Blot, Membrane, Luciferase, Transfection, Expressing, Control, Mutagenesis, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability

doi: 10.3389/fcell.2022.905315

Figure Lengend Snippet: Pharmacological inhibition of CDK9 with AT7519 attenuates LPS-induced pro-inflammatory cytokine release from human MDMs. Panels (A) and (B) IL-6, TNF and IL-10 cytokine release by MDMs untreated (UT) with AT7519 with or without LPS at either 6 h (A) or 24 h (B) . Data represent means ± SEM from n = 6 separate donors, * p < 0.05 ** p < 0.01 *** p < 0.001. (C) IL-1β protein expression by MDMs pre-treated with AT7519 with or without 10 ng/ml LPS (3 h) and 5 mM ATP (1 h). Data represent means ± SEM from n = 3 separate donors, * p < 0.05 ** p < 0.01 *** p < 0.001.

Article Snippet: For CDK9, both 42 and 55 kDa isoforms were probed for using a CDK9 (C12F7) rabbit monoclonal primary antibody (Cell Signaling) and polyclonal goat anti-rabbit secondary antibody (Dako).

Techniques: Inhibition, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability

doi: 10.3389/fcell.2022.905315

Figure Lengend Snippet: Pharmacological inhibition of CDK9 by AT7519 alters markers of pro-inflammatory polarisation. CD80, HLA-DR and CD206 mRNA expression levels by MDMs either untreated (UT) or pre-treated with AT7519 with or without LPS at either 6 (A) or 24 (B) hours. Data represent means ± SEM from n = 6 separate donors, * p < 0.05 ** p < 0.01 *** p < 0.001.

Article Snippet: For CDK9, both 42 and 55 kDa isoforms were probed for using a CDK9 (C12F7) rabbit monoclonal primary antibody (Cell Signaling) and polyclonal goat anti-rabbit secondary antibody (Dako).

Techniques: Inhibition, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability

doi: 10.3389/fcell.2022.905315

Figure Lengend Snippet: Genetic inhibition of CDK9 attenuates LPS-induced pro-inflammatory cytokine release from human MDMs. (A) Western blot of CDK9 protein expression in untreated (UT), lipofectamine-only treated, CDK9 siRNA and control siRNA treated MDMs, compared to Beta-actin as a loading control. Endogenous CDK9 is detected in MDMs as two isoforms of 42 and 55 kDa. (B) The relative expression density of CDK9 in UT versus CDK9 siRNA treated cells as measured by densitometry. (C) Cytotoxicity in control and CDK9 siRNA-treated MDMs as measured by LDH cytotoxicity assay. (D) IL-6 and TNF cytokine release from CDK9 siRNA treated MDMs stimulated with LPS for 24 h compared to controls. (E) Representative flow cytometry plots showing efferocytosis of pHrodo labelled apoptotic neutrophils in CDK9 siRNA-treated compared to control siRNA-treated MDMs. (F) Collated levels of efferocytosis from all donors tested, comparing control siRNA-treated and CDK9 siRNA-treated MDMs. Data represent means ± SEM from n = 6 separate donors, * p < 0.05 ** p < 0.01 *** p < 0.001. ns = no significant difference.

Article Snippet: For CDK9, both 42 and 55 kDa isoforms were probed for using a CDK9 (C12F7) rabbit monoclonal primary antibody (Cell Signaling) and polyclonal goat anti-rabbit secondary antibody (Dako).

Techniques: Inhibition, Western Blot, Expressing, Control, LDH Cytotoxicity Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability

doi: 10.3389/fcell.2022.905315

Figure Lengend Snippet: Genetic over-expression of CDK9 augments LPS-induced pro-inflammatory cytokine release from THP-1 cells. (A) CDK9 mRNA expression in THP-1 cells in either untreated (UT), GFP control and CDK9 plasmid transfected THP-1 cells, assayed by Real Time PCR. (B–D) Cytokine release determined by ELISA, comparing release of IL-6 (B) , TNF (C) and IL-1β (D) cytokines by untransfected (UT), GFP control and CDK9 plasmid transfected cells in response to 6 h stimulation with 1 μg/ml LPS. Data represent means ± SEM from n = 3 experiments, * p < 0.05 ** p < 0.01 *** p < 0.001.

Article Snippet: For CDK9, both 42 and 55 kDa isoforms were probed for using a CDK9 (C12F7) rabbit monoclonal primary antibody (Cell Signaling) and polyclonal goat anti-rabbit secondary antibody (Dako).

Techniques: Over Expression, Expressing, Control, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of Cyclin-Dependent Kinase 9 Downregulates Cytokine Production Without Detrimentally Affecting Human Monocyte-Derived Macrophage Viability

doi: 10.3389/fcell.2022.905315

Figure Lengend Snippet: Summary diagram of effects of CDK9 inhibition and activation on inflammatory pathways in monocyte-derived macrophages. Activation of CDK9 initiates binding with regulatory subunit cyclin T to form the heterodimeric complex P-TEFb, which in turn regulates transcription elongation via phosphorylation of RNA pol II and subsequent mRNA synthesis of anti-apoptotic proteins and inflammatory cytokines. Inhibition of CDK9 in MDMs does not drive caspase-dependent apoptosis despite Mcl-1 downregulation, in contrast to its effects in neutrophils, as MDMs possess additional anti-apoptotic proteins such as Bcl-2 and Bcl-xL . Inhibition of CDK9 can also reduce the release of potent inflammatory cytokines, including TNF, IL-6, and IL-1β after MDM stimulation with LPS, but does not impair MDM efferocytosis of apoptotic neutrophils, nor does it polarize MDMs to a pro- or anti-inflammatory phenotypes, based on cell surface markers.

Article Snippet: For CDK9, both 42 and 55 kDa isoforms were probed for using a CDK9 (C12F7) rabbit monoclonal primary antibody (Cell Signaling) and polyclonal goat anti-rabbit secondary antibody (Dako).

Techniques: Inhibition, Activation Assay, Derivative Assay, Binding Assay, Phospho-proteomics