Journal: Communications Biology

Article Title: Stabilization of CDK6 by ribosomal protein uS7, a target protein of the natural product fucoxanthinol

doi: 10.1038/s42003-022-03522-6

Figure Lengend Snippet: a HT-29 cells were transfected with siCtrl or siuS7 #2. After 18 h, cells were treated with 20 μg/ml cycloheximide and lysed at the indicated times. The expression levels of CDK2, 4, and 6 were analyzed by western blotting. The sample, designated as “0 h half”, is identical to half the amount of the sample at 0 h. b The expression levels of CDK2, 4, and 6 at each time point were quantified. The expression level at 0 h was defined as 100%. c HT-29 cells were transfected with siCtrl, siuS7 #1, or siuS7 #2. After 24 h, cells were incubated with 10 μM MG-132 for 24 h, lysed, and subjected to western blotting. The signal of each western blot was quantified using ImageJ software and normalized by the value of β-actin. The value of each signal was indicated below the blot. d HT-29 cells were lysed, and the endogenous immunoprecipitation assay was performed with anti-CDK2, CDK4, CDK6, cyclin D, and cyclin E antibodies, respectively. Ribosomal proteins uS7 and uS9 in immunoprecipitates were detected by western blotting. e A GST pull-down assay was performed by incubating the recombinant Myc-DDK-tagged CDK6 protein with GST or GST-fused uS7 (GST-uS7)-bound beads. The CDK6 protein that bound to these beads was detected by western blotting.

Article Snippet: GST or the GST-uS7 protein was immobilized onto Glutathione Sepharose 4B beads (GE Healthcare) by incubating the Escherichia coli lysates expressing each protein with the beads at 4 °C for 2 h. The recombinant Myc-DDK-tagged CDK6 protein (OriGene Technologies) was then incubated with GST or GST-uS7-immobilized beads at 4 °C overnight.

Techniques: Transfection, Expressing, Western Blot, Incubation, Software, Immunoprecipitation, Pull Down Assay, Recombinant

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet: CDK4/6 phosphorylate RPRM at serine 98, but the unphosphorylation status of RPRM is critical for its stabilization and nuclear translocation (A and B) Mass spectrometry analysis of the phosphorylation site of RPRM corresponding to CDK4 (A) and CDK6 (B). (C) Time-course analysis of the changes in the RPRM levels of H460-RPRM cells after treated with different inhibitors. (D) Diagrams of full-length RPRM protein and different mutants. (E) Representative immunofluorescence images of the RPRM −/− MEFs exogenously expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) at 30 min after 20 Gy X-irradiation. Scale bars, 10 μm. (F) Co-IP assay confirmed an interaction between ATM and both RPRM-S98A and ΔRPRM (79–109) in H460 cells. (G and H) Change in the ATM levels of H460-NC/RPRM/S98A cells at different times after 2 Gy X-irradiation. (I) Change in the ATM expression of H460-NC/RPRM/S998A cells after cisplatin (CDDP, 15 μM) treatment for 24 h. (J) Relative plating efficiency of H460-NC/RPRM/S98A cells irradiated with 4 Gy X-rays. (K) Exogenous expression of both RPRM and RPRM-S98A increased micronucleus formation in H460 cells upon 2 Gy X-rays. (L) Relative plating efficiency of H460-NC/RPRM/S98A cells treated with 15 μM CDDP. (M) Micronucleus formation of RPRM −/− MEFs expressing full-length RPRM, RPRM-S98A and ΔRPRM (79–109) after exposed to 5 Gy X-irradiation. Data shown represent the means (±SEM) of three biological replicates, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Significance was determined by unpaired two-sample t test.

Article Snippet: Purified recombinant CDK6 , Origene , TP327978.

Techniques: Translocation Assay, Mass Spectrometry, Phospho-proteomics, Immunofluorescence, Expressing, Irradiation, Co-Immunoprecipitation Assay

Journal: iScience

Article Title: RPRM negatively regulates ATM levels through its nuclear translocation on irradiation mediated by CDK4/6 and IPO11

doi: 10.1016/j.isci.2022.105115

Figure Lengend Snippet:

Article Snippet: Purified recombinant CDK6 , Origene , TP327978.

Techniques: Control, Virus, shRNA, Recombinant, Purification, Sequencing, Dominant Negative Mutation, Mutagenesis, Software

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Heatmap analysis of a high-throughput cell cycle protein ELISA of ECs isolated from livers of P6 BMP9/10ib mice vs. PBS control littermates. Data represent n=2 independent analyses (n represents one litter of pups combined for each condition; PBS, n=6 mice; BMP9/10ib, n=7 mice). ( B ) qPCR analysis of ECs isolated from livers of P6 PBS and BMP9/10ib mice (n=5/condition). Data represent mean ± SEM, Mann-Whitney test ( Cdk2 ), and unpaired t-test ( Cdk4 and Cdk6 ). ( C ) Flow cytometry quantification of p-RB1 fluorescence intensity in ECs isolated from livers of P6 PBS and BMP9/10ib mice (n=3/condition). Data represent mean ± SEM, unpaired t-test. *P < 0.05. ( D and E ) Representative IF staining of p-RB1 (green) and IB4 (red) in the peri-optic nerve and mid-plexus regions ( D ) and corresponding quantification of the peri-optic nerve region ( E ) of retinas from vehicle-treated PBS (n=6), palbociclib-treated PBS (n=5), vehicle-treated BMP9/10ib (n=6), and palbociclib-treated BMP9/10ib (n=6) mice. Data in ( E ) represent individual retinas and mean ± SEM, one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant; *P < 0.05, **P < 0.01. Scale bars in ( D ), 50 μm. ( F ) Representative H&E and IHC staining of p-RB1 in 4 μm skin sections of HHT2 patients. Te, telangiectasia; N, normal vessel; Ba, basilar cells.

Article Snippet: Eng f/f , and Cdk6 f/f mice were crossed with Cdh5 -Cre ERT2 mice [Taconic, #13073; ] to generate EC-specific, tamoxifen-inducible KO mice ( Eng iECKO and Cdk6 iECKO ).

Techniques: High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Isolation, Control, MANN-WHITNEY, Flow Cytometry, Fluorescence, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: CDK6-mediated endothelial cell cycle acceleration drives arteriovenous malformations in hereditary hemorrhagic telangiectasia

doi: 10.1101/2023.09.15.554413

Figure Lengend Snippet: ( A ) Representative staining using IB4 (green) and of SMA (red) in whole petals (first two rows), the vein front (third row), and peri-optic nerve (forth row) and mid-plexus (last two rows) regions of retinas from Cdk6 f/f controls and Cdk6 iECKO mice challenged or not (PBS) with BMP9/10ib. Arrows denote AVMs; a, artery; v, vein. Scale bars, 1 mm (whole petal images), 100 μm (vein front), and 50 μm (mid-plexus and peri-optic nerve areas). ( B-D ) Scatter plots showing retinal AVM number ( B ), vein diameter ( C ), and mid-plexus vascular density ( D ) in Cdk6 f/f ;BMP9/10ib controls (n=11-14) and Cdk6 iECKO ;BMP9/10ib (n=8-12) mice. Data represent individual retinas and mean ± SEM, unpaired t-test. **P 0.01, ***P 0.001. ( D ) Schematic illustration of the proposed mechanism of control of the cell cycle in ECs by ALK1 signaling and its relevance for HHT pathogenesis.

Article Snippet: Eng f/f , and Cdk6 f/f mice were crossed with Cdh5 -Cre ERT2 mice [Taconic, #13073; ] to generate EC-specific, tamoxifen-inducible KO mice ( Eng iECKO and Cdk6 iECKO ).

Techniques: Staining, Control

Journal: Cell reports

Article Title: Proteasome activity maintains cell-type-specific gene expression

doi: 10.1016/j.celrep.2026.116973

Figure Lengend Snippet: (A) Replot of , proteins with statistically different changes in protein expression are colored by whether they are tissue-specific (red), not tissue-specific (blue), or not present in the RNA-seq database used for tissue-specificity determination (NA, not available, black). (B) Heatmap of the normalized transcript per million (nTPM) values across 50 different tissue types for the most significantly affected proteins (|log 2 | ≥ 2) in (A). For each protein, nTPM values were normalized to the highest nTPM value across all tissues. Color scale indicates the normalized nTPM values, with white representing lower and red representing higher values as indicated. (C–E) Immunoblots of lysate from HCT116 WT and hRpn10 VWA detecting PALM3, SUSD2, CDK6, SEC31A, RAB25, S100A14, hRpn10, and β-actin. (F) Model of proteasome activity contributing to cell identity that is represented by colored spots inside squares. A cartoon of a cell with the cytosol (yellow) and nucleus (light yellow) is displayed and an aberrant proteome (represented by little color variance) for cells with hRpn10 VWA proteasomes contrasted with a healthy proteome for cells with WT proteasomes. See also and .

Article Snippet: TaqMan probe for CDK6 , Applied Biosystem , Hs01026371_m1.

Techniques: Expressing, RNA Sequencing, Western Blot, Activity Assay