Journal: NPJ Precision Oncology

Article Title: Co-targeting CDK2 and CDK4/6 overcomes resistance to aromatase and CDK4/6 inhibitors in ER+ breast cancer

doi: 10.1038/s41698-022-00311-6

Figure Lengend Snippet: a Evaluation of cell growth of letrozole-resistant (LetR) cells and the sensitive MCF7/S0.5 cell line by crystal violet colorimetric assay. Relative cell growth (%) of independent experiments in triplicates ± SD is shown. Statistically significant differences by two-way ANOVA are shown as **** p ≤ 0.0001. b Quantitative RT-PCR verifying the gene expression alteration of CDK6 , CDK2 , and CCNE1 (encodes cyclin E1). The expression was normalized using PUM1 gene and shown as relative expression in LetR vs. MCF7/S0.5 cells. c Quantitative RT-PCR copy-number assay using Taqman copy-number variants (CNV) primers. Average calculated copy-number values are plotted with bars representing values from replicate measurements ( n = 4) ± SD. d Western blotting analysis of lysates from LetR and MCF7/S0.5 cells cultured at aromatase-dependent growth condition (10% NCS + 100 nM testosterone) in the presence or absence of letrozole (Let, 1 μM). Lysates from AI-resistant MM134-LTED cells and the sensitive MM134 cells treated with or without estradiol (E2, 1 μg/ml) for 4 days. GAPDH was used as loading control. A representative of two biological replicates is shown. e Micrographs of immunocytochemistry analysis of formalin-fixed paraffin-embedded (FFPE) AI-resistant or combined CDK4/6i and fulvestrant-resistant cells stained for CDK6, p-CDK2, and cyclin E1 (black scale bar: 100 µm, gray scale bar: 50 µm). f Western blotting analysis of lysates from the combined palbociclib and fulvetsrant-resistant cells (MPF-R and TPF-R) and the sensitive cell lines (MS and T47D-S, respectively) treated with or without CDK4/6i palbociclib (Pal, 150 nM) and fulvestrant (Ful, 100 nM) for 4 days. β-actin was used as loading control. A representative of two biological replicates is shown. Statistically significant differences calculated by one-way ANOVA are shown as ns p > 0.05, * p ≤ 0.05, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: The Taqman copy-number variant primers used were: Hs01548017_cn (location Chr.7:92606172 on GRCh38, amplicon length 101) for CDK6 , Hs01055552_cn (location Chr.12:55966993 on GRCh38, amplicon length 111) for CDK2 , and Hs07158517_cn (location Chr.19:29819809 on GRCh38, amplicon length 91) for CCNE1 (encodes cyclin E1) (ThermoFisher Scientific).

Techniques: Colorimetric Assay, Quantitative RT-PCR, Gene Expression, Expressing, Western Blot, Cell Culture, Control, Immunocytochemistry, Formalin-fixed Paraffin-Embedded, Staining

Journal: NPJ Precision Oncology

Article Title: Co-targeting CDK2 and CDK4/6 overcomes resistance to aromatase and CDK4/6 inhibitors in ER+ breast cancer

doi: 10.1038/s41698-022-00311-6

Figure Lengend Snippet: The effect of CDK2 or CDK6 silencing in letrozole-resistant (LetR) and parental MCF7/S0.5 cells transfected with two different CDK6-specific (CDK6_5 and CDK6_6), CDK2-specific (CDK2_5 and CDK2_6) or scrambled (control) siRNAs. a Quantitative RT-PCR and western blotting verifying reduction of CDK6 at mRNA (48 h) and protein levels (96 h) post-transfection with CDK6-specific siRNAs. The expression was normalized using PUM1 gene, while β-actin was used as protein loading control. b Cell growth in the presence or absence of letrozole 96 h post-transfection with CDK6-specific siRNAs, as assessed by crystal violet assay. c Quantitative RT-PCR and western blotting verifying reduction of CDK2 at mRNA (48 h) and protein levels (96 h) post-transfection with CDK2-specific siRNAs. The expression was normalized using PUM1 gene and β-actin was used as protein loading control. d Cell growth in the presence or absence of letrozole at 96 h post-transfection, as assessed by crystal violet assay. e Cell growth in the presence of letrozole at different time points following CDK2-specific siRNA transfection, as assessed by crystal violet assay. f Western blotting analysis of G1/S transition cooperative cascades at 96 h after CDK2-siRNA transfection in the absence of letrozole. β-actin was used as loading control. The cell growth is represented as average absorbance at 570 nm of triplicates mean ± SD. Statistically significant differences calculated by two-way ANOVA are shown as ns p > 0.05, * p ≤ 0.05, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: The Taqman copy-number variant primers used were: Hs01548017_cn (location Chr.7:92606172 on GRCh38, amplicon length 101) for CDK6 , Hs01055552_cn (location Chr.12:55966993 on GRCh38, amplicon length 111) for CDK2 , and Hs07158517_cn (location Chr.19:29819809 on GRCh38, amplicon length 91) for CCNE1 (encodes cyclin E1) (ThermoFisher Scientific).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing, Crystal Violet Assay

Journal: NPJ Precision Oncology

Article Title: Co-targeting CDK2 and CDK4/6 overcomes resistance to aromatase and CDK4/6 inhibitors in ER+ breast cancer

doi: 10.1038/s41698-022-00311-6

Figure Lengend Snippet: Kaplan–Meier plots evaluating PFS according to a combined score based on the levels of three biomarkers CDK6, p-CDK2, and cyclin E1, in ER+ metastatic lesions from BC patients in the advanced setting. a Cohort of patients receiving AI-monotherapy. b Cohort of patients treated with combined CDK4/6i and ET. The cutoff values used were: CDK6 H -score > 0, p-CDK2 H -score ≥ 75, and cyclin E1 H -score ≥ 100. A two-sided p -value calculated using log-rank testing is shown. c Representative micrographs of immunohistochemistry analysis of all breast cancer metastasis sections showing low or high CDK6, p-CDK2 and cyclin E1 expression (black scale bar: 100 µm, gray scale bar: 50 µm).

Article Snippet: The Taqman copy-number variant primers used were: Hs01548017_cn (location Chr.7:92606172 on GRCh38, amplicon length 101) for CDK6 , Hs01055552_cn (location Chr.12:55966993 on GRCh38, amplicon length 111) for CDK2 , and Hs07158517_cn (location Chr.19:29819809 on GRCh38, amplicon length 91) for CCNE1 (encodes cyclin E1) (ThermoFisher Scientific).

Techniques: Immunohistochemistry, Expressing

Journal:

Article Title: Human HTm4 is a hematopoietic cell cycle regulator

doi: 10.1172/JCI14025

Figure Lengend Snippet: (a) HTm4 binds to KAP-CDK2 under normal physiological conditions. Samples analyzed are shown on the top. The lane marked anti-CDK2 includes the samples immunoprecipitated with anti-CDK2 mAb and analyzed by Western blot technique using Ab’s as indicated on the left. (b) The C-terminal region of HTm4 is required for its binding to KAP-CDK2. HTm4-Ct, HTm4 without the last 22 amino acids. Ab’s used for the Western blot analysis, after immunoprecipitation with anti-Flag, are listed on the left. The induction of protein expression in the absence of Dox is marked as +, and no induction in the presence of Dox is marked as –. (c) The C-terminal region of HTm4 is required for the enhancement of the phosphatase activity of KAP. The descriptions for c are the same as Figure ​Figure4b,4b, except the top panels show the immunoprecipitation with anti-Flag Ab and the bottom panels are derived from 50 μg of total lysate per sample. The upper bands in the lower panels are the dephosphorylated form of CDK2, marked as de-(P), and the lower bands are the phosphorylated (P) form. A reduction in intensity can be seen in the phosphorylated CDK2 in the presence of overexpressed Flag-HTm4. Representative figures of at least five experiments.

Article Snippet: Primary mAb’s used were anti-KAP, anti-CDK2, both from BD Transduction Laboratories (San Diego, California, USA), anti-HA from Covance Research Products Inc. (Richmond, California, USA), and anti-Flag from Sigma Chemical Co.

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Expressing, Activity Assay, Derivative Assay

Journal:

Article Title: Human HTm4 is a hematopoietic cell cycle regulator

doi: 10.1172/JCI14025

Figure Lengend Snippet: Immunoperoxidase staining of secondary follicles of the tonsil with CDK2, HTm4, KAP, and Ki-67. Sections of reactive tonsillar tissue are stained for expression of (a) CDK2, (b) HTm4, (c) KAP, and (d) Ki-67. Note the strong staining of the proliferating cells of the germinal center for all Ab’s with absence of staining of the surrounding mantle zones. The cellular protein localization is predominantly nuclear for CDK2 and Ki-67 (a and d, respectively) with nuclear and paranuclear staining noted for HTm4 and KAP (b and c, respectively). Magnification ×400, HRP stain; hematoxylin was used as counterstain.

Article Snippet: Primary mAb’s used were anti-KAP, anti-CDK2, both from BD Transduction Laboratories (San Diego, California, USA), anti-HA from Covance Research Products Inc. (Richmond, California, USA), and anti-Flag from Sigma Chemical Co.

Techniques: Immunoperoxidase Staining, Staining, Expressing

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: CDK inhibitors regulate the expression of late genes during EBV lytic replication. (A) qPCR of viral RNAs (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors at 48 h after the induction of lytic replication. Cells were transfected with the BZLF1 expression plasmid and were treated with CDK 2/9 inhibitor (CDK2/9i; 500 nM) or alsterpaullone, 2-cyanoethyl (A2CE; 1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01; n.s., not significant. (B) qPCR analyses of viral RNA levels (center) and viral replication levels (right) in HEK293/EBV(WT) cells treated with CDK inhibitors 48 h after the induction of lytic replication. Cells were transfected with a BZLF1-expressing plasmid and were treated with CDK2/9i (500 nM) or A2CE (1 μM) as indicated on the schedule (left). Results shown are means ± SDs from three independent experiments. Double asterisks, P < 0.01. (C and D) HEK293/EBV(WT) cells were transfected with a TATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and a BZLF1 expression plasmid as indicated. DMSO or a CDK inhibitor was added to the medium at 24 h p.t. (C) Lysates harvested from the cells at 48 h p.t. were subjected to immunoblot analysis with the indicated antibodies. BZLF1 is an IE gene; BMRF1 and BALF2 are E genes; and BRRF2, BALF4 (gB), and BKRF4 are L genes (11, 48, 55, 56). (D) Lysates were also analyzed using luciferase reporter assays. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity). Double asterisks, P < 0.01. (E) HEK293/EBV(WT) cells were first transfected with BZLF1 expression vectors and then treated with CDK inhibitors. The virus yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments and are relative to the virus yield of BZLF1 with DMSO treatment (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay, Virus, Infection

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: CDK2 is responsible for the phosphorylation-mediated stabilization of BDLF4. (A) The cell cycle of HEK293/FLAG-BDLF4 cells was synchronized using a double thymidine block (61). Cells were harvested at the indicated times after release of the block and were analyzed by immunoblotting using the indicated antibodies. AS, asynchronized; h.p.r., hours postrelease. (B and C) HEK293/FLAG-BDLF4 cells were transfected with plasmids expressing shRNA targeting CDK1 or CDK2 mRNA. Cells were lysed at 48 h p.t. for immunoblotting using the indicated antibodies. (D) HEK293/FLAG-BDLF4 cells were transfected with FLAG-BZLF1 and/or shRNA expression plasmids as indicated, followed by CHX treatment (50 mg/ml). The protein levels of FLAG-BDLF4 and FLAG-BZLF1 (control) were detected using anti-FLAG antibodies. Results are presented as means ± SDs from three independent experiments and are shown relative to the protein levels in the absence of CHX treatment. Asterisks, P < 0.05; n.s., not significant. (E) HEK293/FLAG-BDLF4 cells transfected with shCDK2 were treated with MG132 for 24 h before harvesting. Lysates were analyzed by immunoblotting using the indicated antibodies. (F) (Left) Lytic replication was induced by transfection of 293/EBV(WT) cells with a BZLF1-expressing plasmid, followed by transfection with plasmids carrying shCDK2 at 8 h p.t.; the cells were harvested at 48 h p.t. (Right) Lysates were subjected to immunoblotting using the indicated antibodies.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Blocking Assay, Western Blot, Transfection, Expressing, shRNA, Control, Plasmid Preparation

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: Phosphorylation of BDLF4 by CDK2 complexes enhances L gene expression. (A) In vitro phosphorylation assay showing that BDLF4 protein was phosphorylated by recombinant Cyc A1/CDK2 and Cyc E1/CDK2, but not by Cyc B1/CDK1. Histone H1 served as the control. The phosphorylation assay used ATP-γ-S as the phosphodonor and an anti-thiophosphate ester (anti-ThioP) antibody to detect substrate phosphorylation. Immunoblotting (IB) using an anti-BDLF4 or anti-histone H1 antibody shows the levels of the recombinant protein used for the assay. (B) List of the serine/threonine residues in BDLF4 phosphorylated by Cyc A1/CDK2 and Cyc E1/CDK2. These residues were identified by LC–MS-MS analysis. (C) Schematic representation of the S/T sites mutated in the A×6 and A×4 mutants. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid together with pSV40-Rluc as an internal control and BZLF1 and BDLF4 (WT or mutant) expression plasmids as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization against the internal control (Renilla luciferase activity). Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 (WT or mutant) expression plasmids as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Gene Expression, In Vitro, Recombinant, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Transfection, Plasmid Preparation, Mutagenesis, Expressing, Luciferase, Activity Assay, Infection

Journal: Journal of Virology

Article Title: S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus BDLF4 Protein To Temporally Control Late Gene Transcription

doi: 10.1128/JVI.01707-18

Figure Lengend Snippet: Phosphorylation of BDLA4 at T91 plays an important role in the stabilization of BDLF4 protein. (A) Lysates from HEK293 cells transfected with WT BDLF4 or BDLF4 A mutants were analyzed by immunoblotting using anti-FLAG and anti-GAPDH antibodies. (B) HEK293 cells were first transfected with a BDLF4 WT or T91A mutant expression plasmid and then treated with MG132 or BTZMB. Lysates were analyzed by immunoblotting using the indicated antibodies. (C) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT, T91A mutant, or T91E mutant) as indicated. Luciferase activities were measured at 48 h posttransfection. The results are presented as means ± SDs from three independent transfections after normalization to the internal control (Renilla luciferase activity) levels. Lysates were subjected to immunoblotting using the indicated antibodies. Asterisks, P < 0.05. (D) HEK293/EBV(dBDLF4) cells were transfected with the pTATT-oriLyt-Luc reporter plasmid, pSV40-Rluc (internal control), and plasmids expressing BZLF1 and BDLF4 (WT or T91A mutant). Luciferase activities in cells treated with CDK inhibitors (CDK2/9i or A2CE) were assayed. The effects of CDK inhibitors on reporter expression are expressed as percentages of decrease from the level of activity for samples treated with DMSO. The results are means ± SDs from three independent experiments. Asterisks, P < 0.05; double asterisks, P < 0.01, respectively. (E) HEK293/EBV(dBDLF4) cells were transfected with BZLF1 and BDLF4 expression vectors as indicated. The viral yields were determined by counting GFP-positive Akata(–) cells. The results are shown as means ± SDs from three independent experiments relative to the viral yield of BZLF1 plus WT BDLF4 (infectivity value, 1). Double asterisks, P < 0.01.

Article Snippet: Anti-CDK1 and anti-CDK2 rabbit polyclonal antibodies were obtained from Bethyl Laboratories (Montgomery, TX, USA).

Techniques: Phospho-proteomics, Transfection, Western Blot, Mutagenesis, Expressing, Plasmid Preparation, Control, Luciferase, Activity Assay, Infection

Journal: Bioactive Materials

Article Title: Bioengineered platelet nanoplatform enables renal-targeted dexamethasone delivery for chronic nephritis therapy with dual anti-inflammatory/anti-fibrotic effects and minimized systemic toxicity

doi: 10.1016/j.bioactmat.2025.06.002

Figure Lengend Snippet: LN-DEX@PLT ameliorates renal inflammation and reduce podocytes senescence in AD mice. (A–D) Expression of IL-6, TNF-α, FN, Col-I, in the kidney tissue 21 days post-treatment. (E) Schematic diagram of LN-DEX@PLT ameliorates renal inflammation and reduce podocytes senescence. (F) p53 value in the kidney tissue. (G) Immunofluorescence staining of p21. (H) CDK2 value in the kidney tissue. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: Additionally, anti-p53 DINP1, anti-F4/80/ADGRE1, and anti-CDK2 antibodies were procured from Boster Biological Technology, USA.

Techniques: Expressing, Immunofluorescence, Staining