Journal: Respiratory Research

Article Title: Analysis of tumor cell proliferation (Ki-67) and cell cycle regulator proteins in lung adenocarcinoma with different radiological subtypes

doi: 10.1186/s12931-025-03217-6

Figure Lengend Snippet: Cell cycle regulator proteins expression in different CTR groups. A–H the expression of CCNA2, CCNB1, CCND1, TP53, P16, P21, TOP2A, and pRb in lung adenocarcinoma with different CTR. CTR, consolidation-to-tumour ratio

Article Snippet: The antibodies used for the IHC analyses were as follows: CCNB1 (Ab32053, 1:250 dilution; Abcam, MA, USA), P16 (Ab108349, 1:1000 dilution; Abcam, MA, USA), P21 (Ab109520, 1:1000 dilution; Abcam, MA, USA), Cyclin D1 (CCND1) (clone# 5506S, 1:1000 dilution; Cell Signaling Technology, MA, USA), Cyclin A2 (CCNA2) (clone# 67955, 1:1600 dilution; Cell Signaling Technology, MA, USA), TOP2A (12286 T, 1:400 dilution; Cell Signaling Technology, MA, USA), pRb (clone# 8516 T, 1:800 dilution; Cell Signaling Technology, MA, USA), and p53 (Santa Cruz, clone DO1 SC-126, 1:800 dilution; USA).

Techniques: Expressing

Journal: Respiratory Research

Article Title: Analysis of tumor cell proliferation (Ki-67) and cell cycle regulator proteins in lung adenocarcinoma with different radiological subtypes

doi: 10.1186/s12931-025-03217-6

Figure Lengend Snippet: Differences in the positive expression rate of cell cycle regulatory proteins among different CTR groups. A the TMA constructed using clinical samples (HE staining); B the positive expression rates of GGO-LUAD and SN-LUAD on eight cell cycle regulatory proteins (CCNA2, CCNB1, CCND1, P16, P21, TOP2A, TP53, and pRb). CTR consolidation-to-tumour ratio, TMA tissue microarrays

Article Snippet: The antibodies used for the IHC analyses were as follows: CCNB1 (Ab32053, 1:250 dilution; Abcam, MA, USA), P16 (Ab108349, 1:1000 dilution; Abcam, MA, USA), P21 (Ab109520, 1:1000 dilution; Abcam, MA, USA), Cyclin D1 (CCND1) (clone# 5506S, 1:1000 dilution; Cell Signaling Technology, MA, USA), Cyclin A2 (CCNA2) (clone# 67955, 1:1600 dilution; Cell Signaling Technology, MA, USA), TOP2A (12286 T, 1:400 dilution; Cell Signaling Technology, MA, USA), pRb (clone# 8516 T, 1:800 dilution; Cell Signaling Technology, MA, USA), and p53 (Santa Cruz, clone DO1 SC-126, 1:800 dilution; USA).

Techniques: Expressing, Construct, Staining

Journal: Respiratory Research

Article Title: Analysis of tumor cell proliferation (Ki-67) and cell cycle regulator proteins in lung adenocarcinoma with different radiological subtypes

doi: 10.1186/s12931-025-03217-6

Figure Lengend Snippet: Associations between Ki-67 and cell cycle regulator proteins expression. A – D the expression of the following four cell cycle regulator proteins showed a positive correlation with Ki-67 expression, namely CCNA2 , TOP2A, TP53, and pRb, E – H the expression of the remaining four cell cycle regulator proteins (included CCNB1, CCND1, P16 and P21) is not correlated with Ki-67 expression

Article Snippet: The antibodies used for the IHC analyses were as follows: CCNB1 (Ab32053, 1:250 dilution; Abcam, MA, USA), P16 (Ab108349, 1:1000 dilution; Abcam, MA, USA), P21 (Ab109520, 1:1000 dilution; Abcam, MA, USA), Cyclin D1 (CCND1) (clone# 5506S, 1:1000 dilution; Cell Signaling Technology, MA, USA), Cyclin A2 (CCNA2) (clone# 67955, 1:1600 dilution; Cell Signaling Technology, MA, USA), TOP2A (12286 T, 1:400 dilution; Cell Signaling Technology, MA, USA), pRb (clone# 8516 T, 1:800 dilution; Cell Signaling Technology, MA, USA), and p53 (Santa Cruz, clone DO1 SC-126, 1:800 dilution; USA).

Techniques: Expressing

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Cdk1 regulates Cyclin B accumulation in G2 phase (A) Schematic. Cdk1 and Plk1 activities that induce mitosis are detected at the S/G2 border and increase gradually throughout G2 phase. (B) Example of unsynchronized U2OS Cyclin B1-YFP cells growing on micropatterns. Lighter colors indicate higher Cyclin B1-YFP fluorescence. Arrows indicate addition of DMSO or inhibitors. Time lapse 30 min. Scale bar 20 μm. (C) Schematic of the in silico synchronization setup. DMSO-treated cells are synchronized in mitosis (top). The resulting Cyclin B1-YFP fluorescence curve (top right) is used to fit Cyclin B1-YFP fluorescence of individual cells before kinase inhibitor treatment (bottom). (D) Quantification of Cyclin B1-YFP fluorescence of cells growing on micropatterns, treated with DMSO or indicated kinase inhibitors after in silico synchronization as in (B) and (C). Graph shows average and standard error of Cyclin B1-YFP fluorescence (At least 15 cells per condition are showed. Data are representative of 4 additional independent experiments, except for Plk1 inhibitor that is present in 2 additional independent experiments). Please note that only Cyclin B1-YFP fluorescence after kinase inhibitor addition is plotted. (E) Quantification of Cyclin B1, Aurora A, and Aurora B immunofluorescence in 4N U2OS Cdk1as cells after 2 h treatment with 1NMPP1. At least 420 cells per condition are showed. Data are representative of 3 independent experiments. ∗∗∗ p < 0.001, using Student’s t test. Interquartile range and median values are indicated within violin plots.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Fluorescence, In Silico, Immunofluorescence

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Cdk1 regulates transcription of mitotic factors (A) Quantification of Cyclin B1 immunofluorescence in 4N U2OS cells treated with Cdk1 inhibitor (RO3306), cycloheximide, or both for 2 h. The G2 population was separated in silico based on DAPI. At least 520 cells per condition are showed. Data are representative of 2 independent experiments; ∗∗∗ p < 0.001, using ANOVA. Interquartile range and median values are indicated within violin plots. (B) Schematic of the setup for RNA sequencing. HeLa cells were released after double thymidine synchronization. After 4.5 h Cdk1 inhibitor (RO3306) or DMSO was added. After 2 h cells were harvested for RNA sequencing analysis. (C and D) Volcano plots show log2 fold change between treated (RO3306) and non-treated (DMSO) normalized gene expressions (x axis), plotted versus the p value (y axis). Orange circles represent differentially expressed genes, and blue circles represent genes with similar expression (data from 3 independent experiments). (E) Schematic containing a selection of key components involved in direct, inner, and outer feedback regulating Cdk activity. (F) Gene Ontology and p values based on (C).

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Immunofluorescence, In Silico, RNA Sequencing, Expressing, Selection, Activity Assay

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Mathematical model of the cell cycle (A) Schematic representation of the mathematical model. (B) Model prediction (red line) of protein level dynamics after parameter estimation based on quantitative immunofluorescence of indicated proteins in U2OS cells from Akopyan et al. (blue dots). Please note that experimental data are only used until mitotic entry. The decrease of protein levels in mitosis is added to the model to mark mitosis upon full activation of Cdk1. (C) Model estimation of selected cell-cycle activities. Model time refers to number of calculation steps with a fixed duration. (D) Model prediction of Cyclin B level dynamics after inhibition of Cdk1, Cdk2, or Plk1.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Immunofluorescence, Activation Assay, Inhibition

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Mitotic duration after Wee1 inhibition depends on when in G2 phase Wee1 inhibitors are added (A) Model prediction of G2 duration after Wee1 inhibition at different time points in G2 phase. Time when Wee1i added same as in (B) and (C). G2 phase starts approximately at model time 740, and 20 model time corresponds approximately to 30 min. How activities change relative to model time is visible in Figure 3 . (B) Model prediction of accumulated FoxM activity at mitotic entry after Wee1 inhibition at different time points in G2 phase. 100% denotes mitotic levels in absence of Wee1 inhibition. (C) Model prediction of Cyclin B level at mitotic entry after Wee1 inhibition at different time points in G2 phase. 100% denotes mitotic levels in absence of Wee1 inhibition. Striped line indicates apparent minimum Cyclin B levels at mitotic entry. (D and E) U2OS Cyclin B1-YFP cells were monitored by time-lapse microscopy upon addition of Wee1 inhibitors. Three different inhibitors were used: MK1775 (1 μM), PD0166285 (1 μM), and PD407824 (5 μM). (D) Duration of mitosis (x axis) is plotted versus Cyclin B1-YFP level at mitotic entry (y axis). 100% denotes median mitotic levels in absence of Wee1 inhibition. (E) Duration of mitosis (y axis) is plotted versus estimated time before mitosis should Wee1 inhibitors have not been added (x axis). The estimate is based on Cyclin B1-YFP accumulation of control cells in the same experiment ( Figures S3 E and S3F). 31–70 cells per condition are showed. The data are representative of 3 independent experiments.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Inhibition, Activity Assay, Time-lapse Microscopy, Control

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet: Wee1 inhibition in G2 phase leads to a de-coupling of Cdk1 and Plk1 activities (A) Model prediction of Plk1 activity at mitotic entry after Wee1 inhibition at different time points in G2 phase. 100% denotes mitotic levels in absence of Wee1 inhibition. G2 phase starts approximately at model time 740, and 20 model time corresponds approximately to 30 min. How activities change relative to model time is visible in Figure 3 . (B) Model prediction of Cyclin B level, Plk1 level, Plk1 activity, and Cdk1 activity after inhibition of Wee1 at different time points in G2 phase. The dotted line in each graph represents the levels at mitotic entry when Wee1 is not artificially inhibited. Mit indicates mitosis. (C) Flow cytometry analysis of Plk1 levels and Plk1-mediated phosphorylation of TCTP (pTCTP) in mitotic U2OS cells. STLC (10 μM), to block cells in mitosis, was added with or without Wee1i for 2 h before harvest. Graph shows mitotic cells, gated as in Figure S5 A. Wee1i, cells treated with the Wee1 inhibitor MK1775 (1 μM) together with STLC; DMSO, cells treated with DMSO together with STLC. At least 2,000 mitotic cells were quantified per condition in two independent experiments.

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Inhibition, Activity Assay, Flow Cytometry, Phospho-proteomics, Blocking Assay

Journal: iScience

Article Title: Preparation for mitosis requires gradual CDK1 activation

doi: 10.1016/j.isci.2025.112292

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti- Cyclin A2 , Atlas antibodies , Cat# HPA020626, RRID: AB_1847376.

Techniques: Recombinant, Gene Expression, RNA Sequencing, Software

Journal: Pharmaceuticals (Basel, Switzerland)

Article Title: Huafengdan Inhibits Glioblastoma Cell Growth and Mobility by Acting on PLAU and CAV1 Targets.

doi: 10.3390/ph18030428

Figure Lengend Snippet: Figure 1. HFD inhibits proliferation and induces S-phase block and apoptosis in GBM cells in vitro. (a) Changes in cell viability after treatment with different concentrations of HFD (0, 6.25, 12.5, 25, 50, and 100 µg/mL) for 24 h and 48 h were determined using the MTT assay. (b,c) Percentage of EdU-positive cells after treatment of U87 and A172 cells with different concentrations of HFD (0, 25, 50, and 100 µg/mL) for 24 h. (d) Cell cycle distribution of each group of cells after treatment with different concentrations of HFD was determined using flow cytometry. (e) Flow cytometry to determine apoptosis in U87 and A172 cells treated with different concentrations of HFD. (f,g) Western blotting to determine the expression of PCNA, cyclin A2, CDK2, Bax, and Bcl2 proteins. Data are presented as mean ± SD, n = 3, * p < 0.05; ** p < 0.01; *** p < 0.001, compared with the DMSO group. GBM, glioblastoma; HFD, Huafengdan; SD, standard deviation.

Article Snippet: Tetramethyl azole blue (MTT; M8180) was purchased from Beijing Solebaum Biotechnology Co., Ltd. (Beijing, China); an EDU Cell Proliferation Detection Kit (100–121) and Cell Cycle Detection Kit (100–107) were purchased from Albatross Bioscience Co., Ltd. (Guangzhou, Guangdong Province, China); SweScript All-in-One RT SuperMix for qPCR (One-Step gDNA Remover) (G3337) was acquired from Wuhan Xavier Biotechnology Co., Ltd. (Wuhan, Hubei Province, China); Matrigel gel (354234) was obtained from Corning Incorporated (Corning, New York, NY, USA); the stripping solution (FD0050) was purchased from Hangzhou Fode Biotechnology Co., Ltd. (Hangzhou, Zhejiang Province, China); Antibodies against proliferating cell nuclear antigen (PCNA; #13110), Cyclin A2 (#91500), E-cadherin (#3195), Vimentin (#5741), N-cadherin (#13116), MMP2 (#4022), MMP9 (#13667), and β-actin (#4967) were acquired from CST (Shanghai, China); Cyclin-dependent kinase 2 (CDK2; ET1602-6) and protein kinase B (AKT; ET1609-51) were acquired from Hangzhou Hua’an Bio-technology Co., Ltd. (Hangzhou, Zhejiang Province, China); phosphorylated (p)-PI3K (bs-3332R) was obtained from Beijing Boao Sen Biotechnology Co. (Beijing, China); phosphatidylinositol 3-kinase (PI3K; 60225-1-Ig), Urokinase-type plasminogen activator (PLAU; 17968-1-AP), and Caveolin-1 (CAV1; 16447-1-AP) were purchased from Wuhan Sanying Biotechnology Co., Ltd. (Wuhan, Hubei Province, China); phosphorylated (p)-AKT (GB150002) and KI67 (GB121141) were acquired from Wuhan Xavier Bio-technology Ltd. (Wuhan, Hubei Province, China); GAPDH (ET1601-4) was obtained from Guizhou Huayuan Biotechnology Co. (Guiyang, Guizhou, China).

Techniques: Blocking Assay, In Vitro, MTT Assay, Flow Cytometry, Western Blot, Expressing, Standard Deviation