Journal: bioRxiv

Article Title: Proteomic Analysis Reveals Trilaciclib-Induced Senescence

doi: 10.1101/2024.03.12.584620

Figure Lengend Snippet: A) The fitness effects of CRISPR-Cas9 gene knockout on TP53 , CDK4 , CDK6 , and RB1 genes (DepMap Public+Score, Chronos, https://depmap.org/portal/ )) were correlated with trilaciclib sensitivity across 694 cell lines. Pearson correlation coefficient (r) and p-value are shown in each graph. B-C) The sensitivity of trilaciclib varies across different histologic cancer types (DepMap, BRD: BRD-K00003412-300-01-9, PRISM Repurposing Public 23Q2; https://depmap.org/portal/ ). In C) blood cancers are subdivided into Hodgkin lymphoma (HL, 2 cell lines), acute myeloid leukaemia (AML, 21 cell lines), T-cell acute lymphoblastic leukaemia/ lymphoma (T-ALL/LBL, 10 cell lines), myeloproliferative neoplasms (MPN, 12 cell lines), non-Hodgkin lymphoma (NHL, 54 cell lines), and B-cell acute lymphoblastic leukaemia/ lymphoma (B-ALL/LBL, 9 cell lines). D) The inhibition concentration at 50 % cell death (IC50) curves were analysed in K562, JURKAT, U937, MOLT-4, and NCI-H929 cells after 72 hours of treatment. Standard deviation of four biological replicates is shown. E) Percentage of live cells (Annexin V negative and 7-AAD negative cells, light blue), apoptotic cells (Annexin V positive and 7-AAD negative cells, purple), and necrotic cells (7-AAD positive cells, pink) after 72 hours of treatment. The statistical significance of the comparisons with resting is indicated as follows: ‡, P ≤ 0.001; ns, not significant. Standard deviation of three biological replicates is shown.

Article Snippet: Then, it was probed with phospho-Rb (Ser 807/811) (#8516, Cell Signaling), p21 (#2947, Cell Signaling), CDK4 (sc-23896, Santa Cruz), CDK6 (#13331, Cell Signaling), α-Tubulin (T9026) (Sigma-Aldrich) antibodies overnight at 4 °C.

Techniques: CRISPR, Gene Knockout, Inhibition, Concentration Assay, Standard Deviation

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Intrahepatic cholangiocarcinoma escapes from growth inhibitory effect of transforming growth factor-beta1 by overexpression of cyclin D1.

doi: 10.1038/labinvest.3700236

Figure Lengend Snippet: Figure 6 Protein expression of cyclin D1, cyclin D2, cyclin D3, cdk2, cdk4, and cdk6 in cultured MBEC and human ICC cells (CCKS1, HuCCT1, HuH28) with and without TGF-b1 treatment (3.0 ng/ml, 48 h). TGF-b1 inhibits expression of cyclin D1, cdk4 and cdk6 in MBEC. TGF-b1 also inhibits expression of cdk4 (CCKS1, HuCCT1) and cdk6 (CCKS1, HuH28) in ICC cells, while cyclin D1 expression is not influenced by TGF-b1 treatment in ICC cells.

Article Snippet: The membranes were incubated with primary antibodies to cyclin D1 (clone HD11, 1:100, mouse monoclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), cyclin D2 (clone M-20, 1:100, rabbit polyclonal, Santa Cruz Biotechnology, Inc.), cyclin D3 (clone D-7, 1:100, mouse monoclonal, Santa Cruz Biotechnology, Inc.), cdk2 (clone H-298, 1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.), cdk4 (clone H-303, 1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.), cdk6 (clone H-96, 1:200, rabbit polyclonal, Santa Cruz Biotechnology, Inc.), TbR-II (2 mg/ml, goat polyclonal, R&D Systems, Inc.), or b-actin (clone AC-15, 1:5000, mouse polyclonal, Abcam Limited, Cambridge, UK).

Techniques: Expressing, Cell Culture

Journal: Nature Communications

Article Title: DNA framework-engineered chimeras platform enables selectively targeted protein degradation

doi: 10.1038/s41467-023-40244-7

Figure Lengend Snippet: a Schematic illustration of bis-DbTACs design, which is based on DbTACs. b Schematic illustration of three ligand covalent sites of bis-DbTACs equivalent to a DNA tetrahedral scaffold with three polyA domains. Au NPs (5, 10, and 15 nm) correspond to CRBN, CDK9, and CDK6 ligands, respectively. c Cartoon and representative TEM images of bis-DbTACs equivalents. Scale bars are 75 Å and 200 Å, respectively. d WB analysis of the selectively targeted degradation ability of bis-DbTACs at different concentrations and semiquantitative analysis of the grayscale. The error bars indicate the mean ± SD values; n = 3. e Immunofluorescence double-staining images of HepG2 cells treated with/without bis-DbTACs were recorded by confocal laser scanning microscopy. The cell nucleus was stained with DAPI. CDK6 and CDK9 proteins were labeled with anti-CDK6 and anti-CDK9 antibodies, respectively. Scale bars, 10 μm.

Article Snippet: The primary antibody used was mouse CDK6 antibody (Proteintech Group, Rosemont, IL, USA, 66278-1-Ig, 1:100), rabbit CDK9 polyclonal antibody (Proteintech Group, Rosemont, IL, USA, 11705-1-AP, 1:100).

Techniques: Immunofluorescence, Double Staining, Confocal Laser Scanning Microscopy, Staining, Labeling