Journal: bioRxiv

Article Title: CDK2 activity determines the timing of cell-cycle commitment and sequential DNA replication

doi: 10.1101/2020.06.24.169409

Figure Lengend Snippet: (a) Schematics of CDK4/6, CDK2 reporters (left) and APC/C Cdh1 reporter (right). Active CDK4/6 and CDK2 phosphorylate and induce translocation of reporters to the cytoplasm. As APC/C Cdh1 is inactivated, intensity of the reporter starts increasing. (b) Schematic of the CDK4/6 reporter. (c) Top, representative images of CDK4/6, CDK2, and APC/C Cdh1 reporters, Rb (S807/811) immunostaining, and mRNA FISH for E2F1 mRNA in the same MCF-10A cells. Scale bar is 20 µm. Bottom, histograms of CDK4/6, CDK2, and APC/C Cdh1 activities and phosphorylated Rb (p-Rb) at S807/811. Red dotted line indicates a threshold used to classify active CDK4/6 and CDK2, inactive APC/C Cdh1 , and p-Rb populations. Cells were mitogen-released for 14 hr prior to fixation. (d) Scatter plot of the percentage of cells with active CDK4/6, active CDK2, inactive APC/C Cdh1 , and p-Rb at S807/811 in MCF-10A cells as a function of time after mitogen release. E2F1 mRNA level reflects averaged value over time. Solid line is sigmoidal best-fit line. (e) Single-cell correlation of p-Rb at S807/811 versus CDK 4/6 activity (top) and CDK2 activity (bottom) in MCF-10A cells at various time points after mitogen release (8, 10, 12 and 14 hr). Cell density is color-coded. (f) Three-dimensional activity map of CDK4/6 versus CDK2 activity in MCF-10A cells where cell density, the percentage of inactivated APC/C Cdh1 , the percentage of p-Rb at S807/811, and averaged E2F1 mRNA level are color-coded. Black dotted lines represent CDK4/6 activity onset and CDK2 activity onset.

Article Snippet: Anti-phosphor-Rb (S780) (558385) and anti-Rb (554136) were purchased from BD Bioscience, and anti-phophor-Rb (T373) (ab52975) was obtained from Abcam.

Techniques: Translocation Assay, Immunostaining, Activity Assay

Journal: bioRxiv

Article Title: CDK2 activity determines the timing of cell-cycle commitment and sequential DNA replication

doi: 10.1101/2020.06.24.169409

Figure Lengend Snippet: (a) Representative images of multiplexed staining of Rb (S807/811, T373, S608, S780) in MCF-10A cells. Scale bar is 20 µm. (b) Three-dimensional activity map of CDK4/6 versus CDK2 activity where cell density, the percentage of inactivated APC/C Cdh1 , and the percentage of p-Rb at sites S807/811, T373, S608, and S780 are color-coded. MCF-10A cells were mitogen-released for 12 hr prior to fixation. Black dotted lines represent CDK4/6 activity onset and CDK2 activity onset. (c) Top, E2F1 mRNA levels across the distribution of p-Rb at S807/811 in MCF-10A cells. Data are mean ± 95% confidence intervals ( n = 11,741 cells). Bottom, histogram of p-Rb at S807/811. (d) Three-dimensional activity map of CDK4/6 versus CDK2 activity in HLF, HS68, and BJ-5ta cells (top to bottom). Cell density and the percentage of p-Rb at S807/811 are color-coded. Black dotted lines represent CDK4/6 activity onset and CDK2 activity onset. (e) Top, E2F1 mRNA levels across the distribution of p-Rb at S807/811 in HLF cells. Data are mean ± 95% confidence intervals ( n = 18,861 cells). Bottom, histogram of p-Rb at S807/811. (f) Three-dimensional activity maps of CDK4/6 versus CDK2 activity under the control condition, CDK4/6 inhibition, and CDK2 inhibition (top to bottom). The percentage of p-Rb at S807/811, cell density and APC/C Cdh1 are color-coded. After mitogen release for 12 hr, MCF-10A cells were treated with Palbociclib (1 µM) or CDK2i III (60 µM) for 1 hr prior to fixation. Black dotted lines represent CDK4/6 activity onset and CDK2 activity onset.

Article Snippet: Anti-phosphor-Rb (S780) (558385) and anti-Rb (554136) were purchased from BD Bioscience, and anti-phophor-Rb (T373) (ab52975) was obtained from Abcam.

Techniques: Staining, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: CDK2 activity determines the timing of cell-cycle commitment and sequential DNA replication

doi: 10.1101/2020.06.24.169409

Figure Lengend Snippet: (a) Representative time course analysis of CDK4/6 activity, CDK2 activity, and APC/C Cdh1 inactivation labeled with cell-cycle phase and demarcation of CDK activation and APC/C Cdh1 inactivation point. (b) Single-cell traces of CDK4/6 activity aligned to the time of CDK4/6 activation in CDK4/6 high cells. To create more heterogeneity, MCF-10A cells were mitogen-released for various durations (9, 10, and 11 hr) prior to fixation. Orange dots correspond to fixation point. Green dotted line indicates CDK4/6 activity onset. (c) Averaged CDK4/6 activity traces (left) and CDK2 activity traces (right) aligned to CDK4/6 activation in MCF-10A cells. Black, blue, and red lines correspond to all cells, cells with CDK2 activity, and cells without CDK2 activity, respectively. Green dotted line indicates CDK4/6 activity onset. Data are mean ± 95% confidence interval (All population, n = 1,917 cells; with CDK2 activity, n = 879 cells; w/o CDK2 activity, n = 1,038 cells). (d−g) Percentage of p-Rb at sites S807/811 (d), T373 (e), S608 (f), and S780 (g), as a function of CDK4/6 activation in MCF-10A cells. Dotted line indicates percentage of p-Rb in CDK4/6 low cells. Black, blue, and red lines correspond to all cells, cells with CDK2 activity, and cells without CDK2 activity, respectively. (d, All population, n = 1,917 cells; with CDK2 activity, n = 1,038 cells; w/o CDK2 activity, n = 879 cells; CDK4/6 low cells, n = 4,127 cells. e, All population, n = 825 cells; with CDK2 activity, n = 545 cells; w/o CDK2 activity, n = 280 cells; CDK4/6 low cells, n = 2,333 cells. f, g, All population, n = 494 cells; with CDK2 activity, n = 332 cells; w/o CDK2 activity, n = 162 cells; CDK4/6 low cells, n = 1,253 cells). (h) Averaged cyclin E2 protein levels aligned to CDK4/6 activation in MCF-10A cells. Black, blue, and red lines correspond to all cells, cells with CDK2 activity, and cells without CDK2 activity, respectively. Dotted line indicates cyclin E2 protein level in CDK4/6 low cells. Data are mean ± 95% confidence interval (All population, n = 1,930 cells; with CDK2 activity, n = 1,226 cells; w/o CDK2 activity, n = 704 cells; CDK4/6 low cells, n = 3,986 cells). (i) Schematic of TMP-induced rapid expression of cyclin E1. (j) Averaged CDK2 activity traces aligned to the time of CDK4/6 activation for CDK4/6 high cells. Green dotted line indicates CDK4/6 activity onset. Data are mean ± 95% confidence interval (siRNA: Control, n = 3,172 cells; cyclin E1/2, n = 1,042 cells; DHFR-cyclin E1: DMSO, n = 470 cells; TMP, n = 932 cells). (k) Averaged E2F1 mRNA levels aligned to the time of CDK4/6 activation for CDK4/6 high cells. Dotted lines indicate E2F1 mRNA level in CDK4/6 low cells for each treatment. Data are mean ± 95% confidence interval (siRNA: Control, n = 1,699 cells; cyclin E1/2, n = 644 cells; DHFR-cyclin E1: DMSO, n = 233 cells; TMP, n = 552 cells). (j, k) Left, MCF-10A cells were transfected with control (black) or cyclin E1/2 siRNA (red) and were mitogen-released for various durations (9, 10, and 11 hr) prior to fixation. Right, MCF-10A cells expressing DHFR-cyclin E1 were mitogen-released for various durations (9, 10, and 11 hr) with DMSO (black) or TMP (50 µM, red) prior to fixation.

Article Snippet: Anti-phosphor-Rb (S780) (558385) and anti-Rb (554136) were purchased from BD Bioscience, and anti-phophor-Rb (T373) (ab52975) was obtained from Abcam.

Techniques: Activity Assay, Labeling, Activation Assay, Expressing, Transfection

Journal: bioRxiv

Article Title: CDK2 activity determines the timing of cell-cycle commitment and sequential DNA replication

doi: 10.1101/2020.06.24.169409

Figure Lengend Snippet: (a) Percentage of CDK2 low cells for each condition as indicated in MCF-10A cells without and with CDK4/6 inhibitor (palbociclib, 1 µM). Data are mean ± s.d. ( n = 5 biological replicates). (b) Percentages of CDK2 low cells for increasing levels of CDK2 activity at the time of inhibition in MCF-10A cells expressing DHFR-cycE1 without (black) or with TMP (50 µM, red) treatment for 6 hr. Data are mean ± s.d. ( n = 5 biological replicates). (c) Percentage of p-Rb at S807/811 for each treatment condition as indicated for MCF-10A cells without (black) and with DHFR-cyclin E1 expression (green). (d) Scatterplot of Hoechst versus EdU, where cell density is color-coded. Cycling HUVECs expressing DHFR-cyclin E1 were treated with or without TMP (50 µM) for 6 hr followed by EdU (10 µM) treatment for 15 min prior to fixing and staining with Hoechst. (e) Percentage of cells in S phase for cycling HUVECs expressing DHFR-cyclin E1 with or without TMP (50 µM) treatment for 6 hr. Data are mean ± s.d. ( n = 3 biological replicates). P -values were calculated with two-sample t -tests. (f) Histogram of p-Rb at S807/811 for HUVEC cells expressing DHFR-cyclin E1 in G0/G1 phase. Data are mean ± s.d. ( n = 3 biological replicates). Cycling cells were treated with or without TMP (50 µM) for 6 hr followed by acute treatment with CDK4/6 inhibitor (palbociclib, 1 µM) and EdU (10 µM) for 15 min prior to fixation. (g, h) Percentage of p-Rb at S807/811 for each treatment condition as indicated in (g) for Melanocyte, HLF, HS68, BJ-5ta, and RPE1 cells (h) expressing DHFR-cyclin E1. Data are mean ± s.d. ( n = 3 biological replicates). Cycling cells were treated with or without TMP (50 µM) for 6 hr followed by acute treatment with CDK4/6 inhibitor (palbociclib, 1 µM) and EdU (10 µM) for 15 min prior to fixation. P -values were calculated with two-sample t -tests. (i) Schematic of sequential regulation of the Rb/E2F pathway by CDK4/6 and CDK2. Rb phosphorylation by CDK4/6 induces E2F activity and gradual increase in CDK2 activity. High CDK2 activity triggers the commitment point by maintaining Rb phosphorylation, then initiates DNA replication.

Article Snippet: Anti-phosphor-Rb (S780) (558385) and anti-Rb (554136) were purchased from BD Bioscience, and anti-phophor-Rb (T373) (ab52975) was obtained from Abcam.

Techniques: Activity Assay, Inhibition, Expressing, Staining

Journal: Oncology Letters

Article Title: PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis

doi: 10.3892/ol.2014.1957

Figure Lengend Snippet: Cell cycle proteins are overexpressed in human colorectal carcinoma. (A) Western blotting of human colorectal carcinoma tissues (T) and matched adjacent normal colorectal tissues (N) (above the panel) of the expression of the cell cycle proteins (left of the panel). (B) Western blot analysis of the expression of these cell cycle proteins (left of the panel) in eight colorectal carcinoma cell lines (above the panel). The glioblastoma cell line LN229 was included as the positive control and β-actin (Actin) was used as protein loading control. CDK, cyclin-dependent kinases; pRB, phosphorylated retinoblastoma protein.

Article Snippet: Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Positive Control, Control

Journal: Oncology Letters

Article Title: PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis

doi: 10.3892/ol.2014.1957

Figure Lengend Snippet: PD-0332991 blocks RB phosphorylation in colorectal carcinoma cells. (A) The DLD-1 and (B) COLO320 cells were treated at various concentrations of PD-0332991 as indicated (top of the panel) for 48 h. The total proteins were extracted from these treated and untreated cells (controls) and subjected to western blotting using the antibodies as indicated (left of panel). CDK, cyclin-dependent kinases; pRB, phosphorylated retinoblastoma protein.

Article Snippet: Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Techniques: Phospho-proteomics, Western Blot

Journal: Oncology Letters

Article Title: PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis

doi: 10.3892/ol.2014.1957

Figure Lengend Snippet: CDK6 phosphorylates RB for cancer cell growth. (A) COLO320 cells were transduced with lentiviral vectors encoding CDK4 shRNA sequences (CDK4-20, CDK4-64) and (B) CDK6 shRNA sequences (CDK6-893, CDK6-747) and were examined by western blotting using the antibodies as indicated (left of panel). The scrambled shRNA-encoded vector was used as the control (CONT) in the two experiments. (C) The cells transduced with CDK4-shRNA and (D) CDK6-shRNA vector were examined by a cell proliferation assay. The data show the mean ± standard deviation of three independent experiments. Student’s t-test was used to analyze the data. ** P<0.001. CDK, cyclin-dependent kinases; pRB, phosphorylated retinoblastoma protein.

Article Snippet: Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA).

Techniques: Transduction, shRNA, Western Blot, Plasmid Preparation, Control, Proliferation Assay, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Pharmacological inhibition of CDK4/6 impairs diffuse pleural mesothelioma 3D spheroid growth and reduces viability of cisplatin-resistant cells

doi: 10.3389/fonc.2024.1418951

Figure Lengend Snippet: Effects of abemaciclib on Rb protein family. NCI-H28, MSTO-211H, and NCI-H2052 cells were treated according to their respective IC50 value for 48 h with abemaciclib or DMSO as a control. (A) Analysis of phosphorylated levels of RBL2/p130, RBL1/p107, and RB1/p105 proteins were determined by immunoblot. Arrows indicate bands corresponding to phosphorylated RBL1/p107 (S975). GAPDH was used as loading control. (B) Analysis of total levels of RBL2/p130, RBL1/p107, and RB1/p105 proteins determined by immunoblot. Arrows indicate bands corresponding to total RBL2/p130. GAPDH was used as loading control. (C) Levels of total and phosphorylated AKT were determined by immunoblot. GAPDH was used as normalization control. (D) Levels of phpspho-p70S6K (Thr 389), total S6K1 and p27 were determined by immunoblot in MSTO-211H cells upon abemaciclib treatment at its IC50 value. GAPDH was used as normalization control.

Article Snippet: Protein samples were resolved in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes, which were then incubated with antibodies against RBL2/p130 (Abcam, Cambridge, UK; cat# ab183039), phosphoRBL2/p130 S941 (custom by Covalab, Villeurbanne, France), phosphoRBL2/p130 S639 (Santa Cruz, Dallas, TX, USA; cat# sc16301), RBL1/p107 (Cell Signaling Technology, Danvers, MA, USA), phosphoRBL1/p107 S975 (MyBioSource, San Diego, CA, USA; cat# MBS9214765), RB1/p105 (Cell Signaling, cat# 9309), phosphoRB1/p105 S780 (Cell Signaling, cat# 9308), AKT(pan)(C67E7) (Cell Signaling, cat# 4691), phosphoAKT S473 (Cell Signaling, cat# 9271), p27 (Abcam, cat# Ab32034), phospho-p70 S6 kinase (Th389) (Cell Signaling, #9205), S6K1 (Abcam, ab32529), and GAPDH (Santa Cruz, cat# sc-25778).

Techniques: Control, Western Blot

Journal: Scientific Reports

Article Title: Inhibition of MUC1 exerts cell-cycle arrest and telomerase suppression in glioblastoma cells

doi: 10.1038/s41598-020-75457-z

Figure Lengend Snippet: MUC1 Knockdown inhibits the proliferation of glioblastoma cells. ( A ) GFP fluorescence images (magnification, × 100), RT-PCR and western blot (WB) analysis to confirm MUC1 depletion in U373 and T98G with shLuc or sh MUC1 lentivirus infection. GADPH and ACTB were used as an internal control in RT-PCR and western blot, respectively. The blots were cropped and full length blots are presented in Supplementary Fig. S1. ( B ) Cell proliferation assay for 4 days and ( C ) Colony-formation assays with T98G and U373 cells transduced with shLuc or sh MUC1 lentivirus. After 14 days, the cultured cells were fixed with methanol and stained simultaneously with staining solution (0.5% crystal violet in acetic acid/methanol 1:7) and colonies were counted manually. ( B , C ) were representative results from at least 3 independent experiments and were plotted with average of 3 independent experiments. Error bars, SEM. Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against CDKN1B (sc-528), RB1 (sc-50) were from Santa Cruz Biotechnology, GAPDH (2118), phosphorylated RB1 at S780 (p-S780, 9307A), MUC1 (4538) and ACTB (4967) were from Cell Signaling Technology, HRP-conjugated IgGs (111-035-003 and 115-035-003) were from Jackson Immune Research.

Techniques: Knockdown, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Control, Proliferation Assay, Transduction, Cell Culture, Staining