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

Journal: Science signaling

Article Title: HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species

doi: 10.1126/scisignal.aay0482

Figure Lengend Snippet: (A) Ratio of 786-O cells stably infected with a bicistronic lentivirus expressing VHL and Tdtomato (VHL-Tdtomato) to 786-O cells infected with GFP alone (EV-GFP) that had been mixed (1:1) and then treated with 0, 200, or 400 nM palbociclib for 3 to 10 days. Data are means ± SD of n = 4 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA. (B) Immunoblot of total and Ser780-, Ser608-, Ser795-, and Ser807/811-phosphorylated pRb in 786-O cells expressing VHL-Tdtomato or EV-GFP and treated with 100, 200, 400, or 800 nM palbociclib, as indicated by the triangle, for 24 hours. Blots are representative of three biological replicates. (C) 786-O cells that underwent CRISPR/Cas9 editing with an RB1 sgRNA and then were infected, mixed, and treated as in (A). The ratio of RB1-null VHL-Tdtomato cells to RB1-null EV-GFP cells after treatment is shown. Data are means ± SD of n = 3 independent experiments. (D) Immunoblot of Rb, VHL, and actin (loading control) abundance in 786-O cells edited with an RB1 sgRNA (as indicated, +) and infected as described in (C), but not otherwise treated. Blots are representative of three biological replicates. (E) Ratio of 786-O cells stably expressing CDK6(D104S) and either VHL-Tdtomato or EV-GFP that had been mixed and treated as described in (A). Data are means ± SD of n = 3 experiments. (F) Immunoblot of 786-O cells stably infected with lentivirus expressing either WT or mutant (D104S) CDK6 and then treated with 50, 100, 200, 400, 800, or 1600 nM palbociclib, as indicated by the triangle, for 24 hours. Blots are representative of three biological replicates.

Article Snippet: The primary antibodies used were rabbit a-VHL (1:500; Cell Signaling, no. 68547), rabbit α–HIF-2α (1:1000; Bethyl, no. 118-1261), mouse α-vinculin (1:10,000; Sigma, no. V9131), rabbit α-actin (1:2000; Cell Signaling, no. 4970), rabbit α-tubulin (1:1000; Cell Signaling, no. 2146), rabbit α-CDK4 (1:1000; Cell Signaling, #12790), rabbit α-CDK6 (1:1000; Cell Signaling, no. 13331), rabbit α–phospho-pRb (Ser 780 ) (1:5000; Cell Signaling, no. 8180), α–phospho-pRb (Ser 795 ) (used at 1:5000; Cell Signaling, no. 9301), α–phospho-pRb (Ser 608 ) (1:5000; Cell Signaling, no. 8147), ––phospho-pRb (Ser 807/811 ) (1:5000; Cell Signaling, no. 8516), mouse α-pRb (used at 1:5000; Cell Signaling, no. 9309), rabbit α-NDRG1 (1:750; Cell Signaling, no. 5196), and rabbit α–cyclin D1 (1:500; Cell Signaling, no. 2978).

Techniques: Stable Transfection, Infection, Expressing, Western Blot, CRISPR, Control, Mutagenesis