Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) p27 mRNA expression in CLL cells and in healthy B-cells determined by RT-qPCR. (B) Comparison of p27 mRNA expression (determined by RT-qPCR) and p27 protein expression (determined by immunoblot) in the same CLL samples. The black bars show the densitometric quantification of p27 protein levels normalized to actin expression. (C) p27 protein expression in CLL cells and in healthy B-cells (controls). (D) A representative immunoblot showing p27 and actin expression. The arrows mark examples of CLL patient samples with low p27 expression. T, tonsils. (E) Immnofluorescence analysis showing nuclear p27 in CLL cells from four patients. Nuclei are stained with DAPI.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot, Staining

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Myc mRNA expression in CLL cells and in healthy B-cells determined by RT-qPCR. (B) Comparison of Myc mRNA expression (determined by RT-qPCR) and p27 protein expression (determined by immunoblot) in the same CLL samples. The black bars show the densitometric quantification of Myc protein levels normalized to actin expression. (C) Myc protein expression in CLL cells and in healthy B-cells (controls). (D) Representative immunoblot showing Myc and actin expression. T, tonsils

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing p27, Myc and actin expression in cells from CLL patients and from tonsil cells (T, tonsils). (B) Expression levels of Myc and p27 in CLL patients. The protein levels of p27 and Myc as determined by film densitometry and normalized to actin, were classified in three categories with respect to the mean of expression in controls. (C) Classification of patients with low and high Myc protein expression according to their p27 levels.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Immunoblot showing p27 and Myc expression in MEC1 cell line transfected with a p27 expression vector (pCEFL-p27) and the corresponding empty vector. Proteins were analyzed 24 h after transfection. Actin levels are shown to asses protein loading. (B) MEC1 cells were transfected with pEYFPp27 vector and 24 h later the cell cycle of transfected cells was analysed by flow cytometry. The empty vector (pEYFP) was used as controls. The percentage of cells in the G0/G1 phase is indicated. (C) p27 expression rescued fludarabine-induced cell death. Cells were transfected with pEYFPp27 and pEYFP vector and 24 h after transfection the cells were treated with 10 μM fludarabine and cell death was determined 24 h later by trypan blue exclusion assay. (D) MEC1 cells were transfected and treated as in C, and the apoptosis was determined by annexin V binding, assessed by flow cytometry. (E) MEC1 cells were transfected and treated as in C and the levels of active caspase 3 were determined by immunoblot. (F) Correlation of high expression of p27 protein with higher Bcl2 levels in CLL cells.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Flow Cytometry, Trypan Blue Exclusion Assay, Binding Assay

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing the levels of cyclins A and E in CLL cells with and without Myc/p27. Actin levels were also determined to assess protein loading. (B) Correlation between Myc and cyclin A protein levels in CLL cells. (C) Immunoblot showing Myc and p27 protein levels in four patient's cells selected for the experiments shown in D. (D) Molecular filtration chromatographic separation of CLL protein extracts followed by immunoblot for cyclins A, E and Cdk2. The elution of the 55 and 30 kDa proteins is shown at the top. (E) Immunoblot (IB) showing the levels of Myc and p27 in three CLL samples and MEC1 cells and kinase assay of Cdk2 in the same extracts. Proteins were immunoprecipitated with anti Cdk2 and the presence of both cyclin E, p27 and Cdk2 were determined by immunoblot. Lower panel: kinase assays were performed using HisCK − as kinase substrate. M, mock kinase reaction without extract. (F) Immunoprecipitation of Cdk2 and kinase assay of the immunoprecipitates of MEC1 cells and MEC1 cells incubated with lysates from a CLL sample (#01). (G) Immunoprecipitation of Cdk2 and kinase assay from MEC1 cells and from mixed lysates prepared with MEC1 cells and two CLL samples (#01 and #10).

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Filtration, Kinase Assay, Immunoprecipitation, Incubation

Journal: Oncotarget

Article Title: High p27 protein levels in chronic lymphocytic leukemia are associated to low Myc and Skp2 expression, confer resistance to apoptosis and antagonize Myc effects on cell cycle

doi:

Figure Lengend Snippet: (A) Representative immunoblot showing the protein levels of Myc, p27 and Skp2 in CLL cells. Actin levels are also shown as protein loading control. (B) p27 protein levels in CLL cells with high or low expression of Skp2. (C) Myc protein levels in CLL cells with high or low expression of Skp2.

Article Snippet: The antibodies used were anti-Actin (I-19, goat polyclonal, sc-1616), anti-MYC (N-262, sc-764), anti-SKP2 (H-435, sc-7164), anti-p27 (C-19, sc-528 and sc-528-G, rabbit and goat polyclonal respectively) anti cyclin E (M-20, sc-481) or anti-cyclin E (HE12, mouse monoclonal, sc-247), anti-cyclin A (H-432, sc-751), or anti-CDK2 (M2, sc-163) (unless otherwise indicated, all rabbit polyclonals from Santa Cruz Biotechnology), anti-p27 monoclonal antibody (K-25020; Transduction Labs), and anti-Thr(P)-187-p27 (rabbit polyclonal, 71-7700; Invitrogen).

Techniques: Western Blot, Control, Expressing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: NNMT/1-MNA Promote Cell-Cycle Progression of Breast Cancer by Targeting UBC12/Cullin-1-Mediated Degradation of P27 Proteins.

doi: 10.1002/advs.202305907

Figure Lengend Snippet: Figure 2. NNMT promote cell-cycle progression through down-regulating p27 expression. A) Western-blot detection of NNMT in indicated distinct human breast cancer cell lines (SKBR3, MCF7, MDA-MB-231, BT-549, HCC1937, Bcap37, T47D and MDA-MB-468). B) the cell viabilities of above distinct human breast cancer cell lines were tested by Cell-Counting Kit-8 (CCK8) colorimetric assay at indicated time points (0 h, 24 h and 48 h) and relative viable cells were calculated. C) the relative cell viability was analyzed by Cell-Counting Kit-8 (CCK8) colorimetric assay in MDA-MB-231 cells and BT-549 cells transfected with two distinct NNMT specific shRNA (1# and 2#) or control shRNA. D) the relative cell viability of SKBR3 cells transfected with empty

Article Snippet: [26] The anti-β-actin (#4970), anti-p53(#2524), anti-p21(#2947), anti-p57(#2557), anti-CDK2(#2456), anti-CDK4(#12 790), anti-Cyclin E1(#4129), antiCyclin D1(#2978), anti-E2F-1(#3742), and anti-p27 (#3686) were obtained from Cell Signaling Technology (CST, Beverly, Massachusetts, USA).

Techniques: Expressing, Western Blot, Cell Counting, Colorimetric Assay, Transfection, shRNA, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: NNMT/1-MNA Promote Cell-Cycle Progression of Breast Cancer by Targeting UBC12/Cullin-1-Mediated Degradation of P27 Proteins.

doi: 10.1002/advs.202305907

Figure Lengend Snippet: Figure 3. 1-MNA, the metabolite of NNMT, is the key molecule that promotes the progression of breast cancer cell cycle. A,B) Lentivirus mediated over-expression of wide type NNMT(NNMT-wt) or two different site-specific mutated NNMT (NNMT-Y20A and NNMT-D197A) in MCF7 cell lines, then quantification of 1-MNA in cell lysate was determined using LC-MS/MS (A) and protein level of UBC12 and NNMT was determined by western-blot in cell lysates (B). C) flow cytometry analysis of cell-cycle progression of MCF7 cell transfected with lentivirus harboring NNMT-WT, NNMT-Y20A and NNMT-D197A mutants, or control lentivirus, and the results were calculated as percentage of cells in G0/G1 phase, S phase and G2/M phase. D) flow cytometry analysis of cell-cycle progression of SKBR3 cells stimulated with medium or 1-MNA (1 mm, 24 h), and the results were calculated as percentage of cells in G0/G1 phase, S phase and G2/M phase. E) flow cytometry analysis of cell-cycle progression of MDA-MB-231 cells transfected with control shRNA(shNC), shRNA targeting NNMT(shNNMT), and shNNMT plus 1-MNA (1 mm, 24 h), and the results were calculated as percentage of cells in G0/G1 phase, S phase and G2/M phase. F) 10 days after MCF-7 xenograft mouse model was established, the mouse were intraperitoneally injected with 1-MNA (500 mg kg−1) or sterile PBS every 2 days for consecutive two weeks, and tumor volume was calculated according to V = (length × width2)/2. G) MCF7 and SKBR3 cells were stimulated with or without 1-MNA(1.0 mm) for 24 h, then the protein level of p27, p53, and p21 were detected using western-blot. Data are representative of three independent experiments. Data were presented as mean ± SEM. *, P<0.05; **, P<0.001; ***, P<0.001.

Article Snippet: [26] The anti-β-actin (#4970), anti-p53(#2524), anti-p21(#2947), anti-p57(#2557), anti-CDK2(#2456), anti-CDK4(#12 790), anti-Cyclin E1(#4129), antiCyclin D1(#2978), anti-E2F-1(#3742), and anti-p27 (#3686) were obtained from Cell Signaling Technology (CST, Beverly, Massachusetts, USA).

Techniques: Over Expression, Liquid Chromatography with Mass Spectroscopy, Western Blot, Cytometry, Transfection, Control, shRNA, Injection, Sterility

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: NNMT/1-MNA Promote Cell-Cycle Progression of Breast Cancer by Targeting UBC12/Cullin-1-Mediated Degradation of P27 Proteins.

doi: 10.1002/advs.202305907

Figure Lengend Snippet: Figure 4. NNMT/1-MNA promote p27 degradation via increasing cullin-1 neddylation. A) qRT-PCR analysis of p27 mRNA and NNMT mRNA transcrip- tion in indicated cells(MCF7/VECTOR and MCF7/NNMT, SKBR3/VECTOR and SKBR3/NNMT, MDA-MB-231/shNC and MDA-MB-231/shNNMT). B) Western-blot analysis of protein level of p27 and NNMT in NNMT knockdown cells (MDA-MB-231/shNNMT) and C) NNMT over-expression cell line (SKBR3/NNMT) at different time points (0 h, 6 h, 9 h, and 12 h) after CHX (30 μg mL−1) treatments, and its relative expression to 𝛽-actin was calculated. D, MCF7, or MDA-MB-231 transfected with NNMT shRNA (MDA-MB-231/shNNMT)(E) were firstly stimulated with or without 1-MNA(1.0 mm) for 24 h, then treated with CHX (30 μg mL−1) for indicated time point (0 h, 6 h, 9 h, and 12 h), then the protein level of p27 was detected using Western-blot and its relative expression to 𝛽-actin was calculated. F) western-blot analysis of protein level of p27 in MCF7, SKBR3 and MDA-MB-231 cells treated with MLN4924 (600 nm for 24 h) or DMSO as indicated. G, flow cytometry analysis of cell-cycle progression of SKBR3/VECTOR cells, NNMT-over-expressing SKBR3 cells (SKBR3/NNMT), and SKBR3/VECTOR and SKBR3/NNMT cells treated with MLN4924 (600 nm, 24 h), and the results were calculated as percentage of cells in G0/G1 phase, S phase and G2/M phase. H) Western-blot detection of cullin-1 and NNMT in NNMT over-expression cell line (MCF7/NNMT, SKBR3/NNMT) and two distinct NNMT specific shRNA (shNNMT-1 and shNNMT-2) transfected cell line (BT-549 and MDA-MB-231), empty vector or control shRNA transfected cell lines were used as control, and nedd8 conjugated cullin-1 was indicated. I) indicated cells were stimulated with or without 1-MNA(1.0 mm) for 24 h, protein level of cullin-1 was detected using western-blot and nedd8 conjugated cullin-1 was indicated. Data were presented as mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: [26] The anti-β-actin (#4970), anti-p53(#2524), anti-p21(#2947), anti-p57(#2557), anti-CDK2(#2456), anti-CDK4(#12 790), anti-Cyclin E1(#4129), antiCyclin D1(#2978), anti-E2F-1(#3742), and anti-p27 (#3686) were obtained from Cell Signaling Technology (CST, Beverly, Massachusetts, USA).

Techniques: Quantitative RT-PCR, Plasmid Preparation, Western Blot, Knockdown, Over Expression, Expressing, Transfection, shRNA, Cytometry, Control

Journal: Molecular Cancer Therapeutics

Article Title: A Whole-Genome CRISPR Screen Identifies AHR Loss as a Mechanism of Resistance to a PARP7 Inhibitor

doi: 10.1158/1535-7163.mct-21-0841

Figure Lengend Snippet: Figure 6. RBN-2397 induces G1 arrest and apoptosis through AHR activity. A, Schematic of the FastFUCCI cell-cycle reporter system. Red, yellow, and green fluorescent cells correspond to the cells in G1, G1–S and S/G2–M, respectively. B, Cell-cycle analysis of FastFUCCI-labeled NCI-H1373 treated with the indicated drugs for 48 hours. C, RT-qPCR data showing the relative mRNA levels of p27/CDKN1B in parental and AHR knockout cells treated with the indicated drugs for 24 hours. D, Western blots of cell lysates from parental and AHR knockout NCI-H1373 cells treated with the indicated concentrations of RBN-2397 for 48 hours. E, Fraction of parental (top) and AHR knockout (bottom) NCI-H1373 cells staining positive for Annexin V treatment with the indicated drugs for 24 hours. Data shown as mean values SD; n ¼ 3.

Article Snippet: Anti–V5-Tag (CST, 13202); anti–Myc-Tag (CST, 2040S); antiGAPDH (CST, 3683S); anti-p21 Waf1/Cip1 (CST, 2947T); anti-p27 Kip1 (CST, 9313T); anti-Rb (CST, 3686T); anti–Phospho-Rb (Ser807/ 811; CST, 8516S); anti-Histone H3 (CST, 3683); anti–PhosphoHistone H3 (Ser10; CST, 3377S); anti-streptavidin (Thermo Fisher Scientific, S911); anti-Rabbit HRP (horseradish peroxidase) antibody (CST, 7074P2); and anti-Mouse HRP antibody (CST, 7076S).

Techniques: Activity Assay, Cell Cycle Assay, Labeling, Quantitative RT-PCR, Knock-Out, Western Blot, Staining

Journal: Oncogene

Article Title: Induction of S-phase arrest and p21 overexpression by a small molecule 2[[3-(2,3-dichlorophenoxy)propyl] amino]ethanol in correlation with activation of ERK.

doi: 10.1038/sj.onc.1207645

Figure Lengend Snippet: Figure 3 Effect on p21 and p27 expression in DLD-1 cells. (a) Cells were treated with 20-mM of 2,3-DCPE and harvested at various times. Cells treated with DMSO were used as a control for each time point. (b) Cells were treated with different concentration of 2,3-DCPE for 24 h. Cells treated with DMSO for 24 h were used as a control

Article Snippet: Rabbit anti-human p27, ERK, phospho-ERK, p38, phospho-p38, JNK and phospho-JNK, and mouse anti-human p21 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Control, Concentration Assay

Journal: Cell reports

Article Title: Molecular control of cell density-mediated exit to quiescence

doi: 10.1016/j.celrep.2021.109436

Figure Lengend Snippet: (A) Top: schematic of the experimental setup. Center: correlation between nuclear cyclin D1 level in newborn cells and local density in mothers. Pearson correlation coefficient, −0.193, p = 2.6 × 10 −73 . Bottom: correlation between nuclear p27 level in newborn cells and local density in mothers. Pearson correlation coefficient, 0.257, p = 6.2 × 10 −130 . Line plots are population medians in each bin; shaded error bars are 95% confidence intervals (n ≥ 50 cells per bin, n = 8,574 cells total). Data are representative of 4 independent experiments. (B) Top: schematic of the experimental setup. Cyclin D1 overexpression (O.E.) was driven by a doxycycline-inducible promoter. Bottom left: correlation of Rb hyperphosphorylation in newborn cells and local density in mothers (n ≥ 20 cells per bin; control, n = 694 cells total; cyclin D1 O.E., n = 973 cells total). Data are representative of 3 independent experiments. Bottom right: percentage of newborn cells with hyperphosphorylated Rb when gating for cells whose mothers experienced different local densities (n ≥ 25 cells per data point). The same gating was used for control and cyclin D1 O.E. Student’s t test; control conditions, p = 2.9 × 10 −4 ; cyclin D1 O.E. conditions, p = 0.14 (n = 3 independent experiments). (C) Top: schematic of the experimental setup. Bottom left: correlation of Rb hyperphosphorylation in newborn cells and local density in mothers (n ≥ 25 cells per bin; si-Control, n = 1,063 cells total; si-p27, n = 1,622 cells total). Data are representative of 3 independent experiments. Bottom right: percentage of newborn cells with hyperphosphorylated Rb when gating for cells whose mothers experienced different local densities (n ≥ 28 cells per data point). The same gating was used for si-Control and si-p27. Student’s t test; si-Control conditions, p = 0.014; si-p27 conditions, p = 0.15 (n = 3 independent experiments). (D) Cyclin D1/p27 ratio in newborn cells as a function of local cell density in mothers (the ratio is expressed in arbitrary units and not indicative of actual stoichiometry; data from A). Pearson correlation coefficient, −0.423, p < 1 × 10 −130 . Line plots are population medians in each bin; shaded error bars are 95% confidence intervals (n ≥ 50 cells per bin, n = 8,574 cells total). Arrows indicate data points represented by circles in (F). Data are representative of 4 independent experiments. a.u., arbitrary unit. (E) Percentage of newborn cells with Rb hyperphosphorylation as a function of cyclin D1/p27 ratio (n ≥ 20 cells per bin, n = 8,669 total, data from A). The Hill function was fitted onto the linear scale data. Hill coefficient, 6.3 ± 0.9 (mean ± s.t.d. of 4 independent experiments). (F) Percentage of newborn cells with Rb hyperphosphorylation as a function of cyclin D1 and p27 protein level (n ≥ 10 cells per bin, n = 8,255 total, data from A). Data are representative of 4 independent experiments. (G) Percentage of newborn cells with Rb hyperphosphorylation as a function of cyclin D1/p27 ratio after binning cells into quartiles by local density in mothers (n ≥ 20 cells per bin, n ≥ 2,000 total for each quartile, data from A). The Hill curve was fitted to the entire dataset (the same curve as in E). See also .

Article Snippet: Antibodies used in the study: rabbit anti-cyclin D1 (Abcam ab16663, 1:500, validated with knock-down and overexpression), mouse anti-p27 (Cell Signaling Technology 3698, 1:500, validated with knock-down and overexpression), rabbit anti-phospho-Rb (Ser807/811) (Cell Signaling Technology 8516, 1:2500, validated with knock-down and CDK4/6 inhibitor palbociclib treatment), rabbit anti-phospho-Rb (Ser807/811) (Alexa Fluor 488 conjugate) (Cell Signaling Technology 4277, 1:2500, validated with CDK4/6 inhibitor palbociclib treatment), mouse anti-N-cadherin (Santa Cruz Biotechnology sc-393933, 1:250, validated with knock-down), and mouse anti-HA tag (Abcam ab130275, 1:1000, validated with overexpression).

Techniques: Over Expression, Control

Journal: Cell reports

Article Title: Molecular control of cell density-mediated exit to quiescence

doi: 10.1016/j.celrep.2021.109436

Figure Lengend Snippet: (A) Schematic of the ERK signaling pathway in mammalian cells. (B) Top: schematic of the experimental setup. ERK activity was measured by the EKAEV-NLS FRET sensor, which was used throughout unless stated otherwise. Bottom left: sample traces of ERK activity at different local density and with DMSO or MEK inhibitor (PD0325901, 100 nM) treatment. Shaded regions indicate ERK activity above baseline (average activity when treated with the MEK inhibitor). Bottom right: correlation between time-averaged ERK activity and local density. Linear regression slope, −1.09 versus −0.41. Arrows indicate data points represented by sample traces. Line plots are population medians in each bin; shaded error bars are 95% confidence intervals (n ≥ 39 cells per bin; DMSO, n = 11,693 cells total; MEK inhibitor, n = 12,447 cells total). Data are representative of 3 independent experiments. (C) Cell density was acutely increased by plating additional non-fluorescent cells (1.32 × 10 5 cells/cm 2 ). Line plots are population medians of 140 or more cells; shaded error bars are 95% confidence intervals. Student’s t test comparing ERK activity in mock versus treatment: 15 min before treatment, p = 0.21; 30 min after treatment, p = 1.5 × 10 −26 . Data are representative of 3 independent experiments. (D) Left: schematic of the experimental setup. Cell traces are shown on the bottom. Right: ERK activity after releasing cells from confinement, measured by the ERK KTR. Line plots are population medians of 92 or more cells; shaded error bars are SEM. Student’s t test comparing ERK activity in released (periphery) versus unreleased cells: 15 min before release, p = 0.29; 3 h after release, p = 8.0 × 10 −8 . Data are representative of 2 independent experiments. (E) Top: schematic of the experimental setup. Bottom: percentage of newborn cells with Rb hyperphosphorylation after inhibition of the MEK-ERK pathway (MEK inhibitor: PD0325901, 100nM; ERK inhibitor: SCH772984, 1 μM) (n ≥ 785 cells per condition). Student’s t test; MEK inhibitor versus DMSO, p = 6.5 × 10 −5 (n = 3 independent experiments); ERK inhibitor versus DMSO, p = 4.4 × 10 −4 (n = 2 independent experiments). (F) Top: schematic of the experimental setup. Constitutively active MEK1 (MEK1-CA: S218D/S222D) O.E. was driven by a doxycycline-inducible promoter. Center: correlation of Rb hyperphosphorylation in newborn cells and local density in mothers (n ≥ 16 cells per bin; control, n = 2,068 cells total; MEK1-CA O.E., n = 1,984 cells total). Data are representative of 4 independent experiments. Bottom: percentage of newborn cells with hyperphosphorylated Rb when gating for cells whose mothers experienced different local densities(n ≥ 10 cells per data point). The same gating was used for control and MEK1-CA O.E. Student’s t test; control conditions, p = 2.4 × 10 −5 ; MEK1-CA O.E. conditions, p = 0.020; control versus MEK1-CA O.E., high density, p = 0.010 (n = 4 independent experiments). (G) Top: schematic of the experimental setup. Bottom: mRNA and protein levels after drug treatment, measured by RNA FISH and immunofluorescence, respectively (MEK inhibitor: PD0325901, 100 nM). Error bars are 95% confidence intervals. Kolmogorov-Smirnov test; cyclin D1 mRNA, p = 7.9 × 10 −99 ; p27 mRNA, p = 1.6 × 10 −17 ; cyclin D1 protein, p = 1.5 × 10 −59 ; p27 protein, p = 4.6 × 10 −67 (n ≥ 320 cells per condition). Data are representative of 3 independent experiments. See also .

Article Snippet: Antibodies used in the study: rabbit anti-cyclin D1 (Abcam ab16663, 1:500, validated with knock-down and overexpression), mouse anti-p27 (Cell Signaling Technology 3698, 1:500, validated with knock-down and overexpression), rabbit anti-phospho-Rb (Ser807/811) (Cell Signaling Technology 8516, 1:2500, validated with knock-down and CDK4/6 inhibitor palbociclib treatment), rabbit anti-phospho-Rb (Ser807/811) (Alexa Fluor 488 conjugate) (Cell Signaling Technology 4277, 1:2500, validated with CDK4/6 inhibitor palbociclib treatment), mouse anti-N-cadherin (Santa Cruz Biotechnology sc-393933, 1:250, validated with knock-down), and mouse anti-HA tag (Abcam ab130275, 1:1000, validated with overexpression).

Techniques: Activity Assay, Inhibition, Control, Immunofluorescence

Journal: Cell reports

Article Title: Molecular control of cell density-mediated exit to quiescence

doi: 10.1016/j.celrep.2021.109436

Figure Lengend Snippet: (A) ERK activity was time-averaged over 4 h. Top: line plots are population medians of 25 or more cells per bin; shaded error bars are 95% confidence intervals. Bottom: population medians of ERK activity (n ≥ 200 cells per condition). Data are representative of 3 independent experiments. (B) Top: line plots are population medians of 30 or more cells per bin; shaded error bars are 95% confidence intervals. Bottom: population medians of cyclin D1/p27 ratio and percentage of cells with Rb hyperphosphorylation (n ≥ 250 cells per condition). Data are representative of 3 independent experiments. (C) Percentage of newborn cells with Rb hyperphosphorylation is plotted as a function of cyclin D1/p27 ratio (n ≥ 20 cells per bin, n ≥ 320 total for each condition). The Hill curve was fitted to the entire dataset. The color-coded schematic in the legend corresponds to the heatmaps in (B). See also .

Article Snippet: Antibodies used in the study: rabbit anti-cyclin D1 (Abcam ab16663, 1:500, validated with knock-down and overexpression), mouse anti-p27 (Cell Signaling Technology 3698, 1:500, validated with knock-down and overexpression), rabbit anti-phospho-Rb (Ser807/811) (Cell Signaling Technology 8516, 1:2500, validated with knock-down and CDK4/6 inhibitor palbociclib treatment), rabbit anti-phospho-Rb (Ser807/811) (Alexa Fluor 488 conjugate) (Cell Signaling Technology 4277, 1:2500, validated with CDK4/6 inhibitor palbociclib treatment), mouse anti-N-cadherin (Santa Cruz Biotechnology sc-393933, 1:250, validated with knock-down), and mouse anti-HA tag (Abcam ab130275, 1:1000, validated with overexpression).

Techniques: Activity Assay

Journal: Cell reports

Article Title: Molecular control of cell density-mediated exit to quiescence

doi: 10.1016/j.celrep.2021.109436

Figure Lengend Snippet: (A) Model. (B) Schematic of the ultra sensitive response curve of Rb hyperphosphorylation as a function of the cyclin D1/p27 ratio. In this example, the Hill function has EC 50 = 1 (the ratio at which 50% of cells have hyperphosphorylated Rb) and Hill coefficient n H = 6.3 (measured in ). EC 90 /EC 10 = 2.0, EC 95 /EC 5 = 2.5. (C) Cell-to-cell variability in the cyclin D1/p27 ratio in cells that experienced similar local density and mitogen conditions. A histogram of the cyclin D1/p27 ratio in newborn cells when local cell density experienced by mothers is between 0.18 and 0.20 (n = 767 cells, data from ) is overlaid with the Hill function fitted to the entire dataset (data from , n H = 6.3).

Article Snippet: Antibodies used in the study: rabbit anti-cyclin D1 (Abcam ab16663, 1:500, validated with knock-down and overexpression), mouse anti-p27 (Cell Signaling Technology 3698, 1:500, validated with knock-down and overexpression), rabbit anti-phospho-Rb (Ser807/811) (Cell Signaling Technology 8516, 1:2500, validated with knock-down and CDK4/6 inhibitor palbociclib treatment), rabbit anti-phospho-Rb (Ser807/811) (Alexa Fluor 488 conjugate) (Cell Signaling Technology 4277, 1:2500, validated with CDK4/6 inhibitor palbociclib treatment), mouse anti-N-cadherin (Santa Cruz Biotechnology sc-393933, 1:250, validated with knock-down), and mouse anti-HA tag (Abcam ab130275, 1:1000, validated with overexpression).

Techniques:

Journal: Cell reports

Article Title: Molecular control of cell density-mediated exit to quiescence

doi: 10.1016/j.celrep.2021.109436

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used in the study: rabbit anti-cyclin D1 (Abcam ab16663, 1:500, validated with knock-down and overexpression), mouse anti-p27 (Cell Signaling Technology 3698, 1:500, validated with knock-down and overexpression), rabbit anti-phospho-Rb (Ser807/811) (Cell Signaling Technology 8516, 1:2500, validated with knock-down and CDK4/6 inhibitor palbociclib treatment), rabbit anti-phospho-Rb (Ser807/811) (Alexa Fluor 488 conjugate) (Cell Signaling Technology 4277, 1:2500, validated with CDK4/6 inhibitor palbociclib treatment), mouse anti-N-cadherin (Santa Cruz Biotechnology sc-393933, 1:250, validated with knock-down), and mouse anti-HA tag (Abcam ab130275, 1:1000, validated with overexpression).

Techniques: Recombinant, Clinical Proteomics, ISH Cell Assay, SYBR Green Assay, Software, Microscopy, Cell Tracking Assay

Journal: Molecular Medicine

Article Title: p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

doi: 10.1007/bf03401838

Figure Lengend Snippet: Fig. 1. Electron microscopy of p27 in normal lymphocytes. Freshly harvested lymphocytes were processed for immunoelecton microscopy and stained with polyclonal anti-p27 antibodies. Two representative cells are shown. Clusters of p27 associated with the plasma membrane are indicated by arrows. Bar 100 nm.

Article Snippet: The cells were then stained for p27 using either immunoglobulin G (IgG; negative control) or rabbit anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit Cy5 (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Electron Microscopy, Microscopy, Staining, Clinical Proteomics, Membrane

Journal: Molecular Medicine

Article Title: p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

doi: 10.1007/bf03401838

Figure Lengend Snippet: Fig. 2. Western blots of isolated membrane. Lymphocyte- membrane fraction isolated as an S-100 pellet (membrane) and isolated nuclei were stained for p27. They are also stained with fractionation controls, TAFIID (a nuclear transcription factor) and the CD3 T cell surface marker, as indicated.

Article Snippet: The cells were then stained for p27 using either immunoglobulin G (IgG; negative control) or rabbit anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit Cy5 (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Western Blot, Isolation, Membrane, Staining, Fractionation, Marker

Journal: Molecular Medicine

Article Title: p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

doi: 10.1007/bf03401838

Figure Lengend Snippet: Fig. 3. p27 expression in detergent-treated lymphocytes. Freshly harvested peripheral blood lymphocytes were incubated in cold buffer containing 0.5% NP-40 (A) or buffer lacking NP- 40 (B) for 30 min, washed, fixed, permeabilized, and stained in suspension for p27 expression. Confocal images acquired at equal (pmt) sensitivity are shown. Membrane, but not nuclear expression of p27, persists after NP-40.

Article Snippet: The cells were then stained for p27 using either immunoglobulin G (IgG; negative control) or rabbit anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit Cy5 (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Expressing, Incubation, Staining, Suspension, Membrane

Journal: Molecular Medicine

Article Title: p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

doi: 10.1007/bf03401838

Figure Lengend Snippet: Fig. 4. Presence of p27 in detergent-insoluble membrane domains. (A) Insolubility of p27 in NP-40. Western blots analy- sis of nonadherent peripheral blood mononuclear cells (PBMC) or PBMC activated for 24 hr in 100 nM phorbol ester 12-O- tetradecanoylphorbol-13-acetate (PMA) and 1 g/ml phytohemagglutinin (PHA). Expression of proteins in total cell extracts (tot) and in membrane fractions prepared from an equivalent number of cells is shown. “NP-40” indicates the supernatent obtained after 60 min incubation in cold NP-40. The protein content of the insoluble NP-40 pellet was determined by dissolving that pellet in sodium dode- cyl sulfate (SDS) buffer (laemmli). PCNA positivity indicates the activated status of the mitogen-exposed cells and that the nuclear frac- tion is not present in membrane preparations. Molecular weights are indicated in margins. (B) Insoluble p27 can be dissolved in octyl- glucoside. Western blots indicating p27 levels in the total cellular pellet after lysis in hypotonic buffer (lane 1) and in the pellet remaining after cells were incubated for 30 min in cold 1% Triton X-100 (Triton insol., lane 2). The Triton-insoluble pellet subse- quently was dissolved in cold octylglucoside (OG), as described in text, and solubilized p27 (OG-supernatent, lane 3), and remaining insoluble p27 (OG pellet, lane 8) determined. Rapidly migrating variants of p27, as seen in the Triton-soluble fractions (Triton super- natents, lanes 6, 7) have been described elsewhere [Yaroslavskiy et al., submitted, and (51)].

Article Snippet: The cells were then stained for p27 using either immunoglobulin G (IgG; negative control) or rabbit anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit Cy5 (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Membrane, Western Blot, Expressing, Incubation, Lysis

Journal: Molecular Medicine

Article Title: p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

doi: 10.1007/bf03401838

Figure Lengend Snippet: Fig. 5. Colocalization of p27 and CD59 in unstimulated but not in activated lymphocytes. (A) Overlapping of p27 and CD59 ex- pression in Triton X-100 treated lymphocytes. Cells treated with Triton X-100 were stained directly with (PE)-conjugated anti-CD59 and indirectly with anti-p27 as described in the text. Color channels were set so that CD59 appears green, p27 appears red, and overlap appears yellow. (B) Lack of aggregation of p27 in lymphocytes activated by CD59 crosslinking. Nonadherent peripheral blood mononu- clear cells (PBMC) were activated with murine anti-CD59, crosslinking with anti-mouse immunoglobulin G-(IgG-FITC) and incuba- tion at 37C. They subsequently were stained indirectly with p27 as described. p27 staining (red) and with CD59 staining (green) is shown for three representative cells. p27 staining (red) of the cells is shown in the left column. The right column shows the same cells, gated to reveal both p27 (red) and CD59 (green) staining. Control staining is shown below, performed as above, but using an isotype control IgG instead of anti-p27. Staining with isotype control for CD59 (not shown) was negative.

Article Snippet: The cells were then stained for p27 using either immunoglobulin G (IgG; negative control) or rabbit anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit Cy5 (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Staining, Control

Journal: Molecular Medicine

Article Title: p27Kip1 Localizes to Detergent-insoluble Microdomains Within Lymphocyte Membranes

doi: 10.1007/bf03401838

Figure Lengend Snippet: Fig. 6. Patching of CD69, but not of p27, upon mitogen stimulation of lymphocytes. Lymphocytes were incubated for 16 hr in the presence of medium alone (unactivated) or medium containing phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; 100 ng) and phytohemagglutinin (PHA; 1 g/ml), and stained for the lipid analogue DIOC16(3) (green), p27 (blue) or CD69 (red), as described in the text. Areas of overlap of DIOC16(3) and p27 appear as white (overlay in 4th column); overlap of CD69 and DIOC16 (3) are yellow.

Article Snippet: The cells were then stained for p27 using either immunoglobulin G (IgG; negative control) or rabbit anti-p27 (Santa Cruz Biotechnology, Santa Cruz, CA), followed by goat anti-rabbit Cy5 (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Incubation, Staining

Journal: Journal of Biomedical Research

Article Title: Severer nodular lesion in white matter than in gray matter in simian immunodeficiency virus-infected monkey, but not closely correlated with viral infection

doi: 10.7555/JBR.33.20180047

Figure Lengend Snippet: Distribution of p27 positive cells in MNs and PCs in CC, DWM and Ctx.

Article Snippet: Mouse anti-SIV Nef protein (p27, 1:500, Santa Cruz Biotechnology, USA) in combination of anti-Iba-1 was applied to examine SIV-infected microglia and macrophages.

Techniques:

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: (A) The experimental approach. (B) GATA3, LHX1, WT1, and HNF4A expression in RCC cells treated with Dec to deplete DNMT1 or UNC1215 to inhibit L3MBTL3. Dec 0.5 μM or UNC1215 1.0 μM added days 1, 2, and 3 each week to RCC cells ACHN and SKRC45. Gene expression by qRT-PCR relative to vehicle-treated control. Plotted are means ± SD for three independent experiments, *p < 0.01, two-sided t test drug versus vehicle. (C) Western blot for MYC (master regulator of proliferation) and p27/CDKN1B (mediates cell-cycle exits by differentiation). Lanes were run on the same gel but were non-contiguous as indicated. (D) Giemsa-stained cytospin preparations of cells harvested on day 5. Scale bar (white line), 12.5 μM; magnification, 400×. (E) Cell counts by automated counter. Plotted are means ± SD from three independent experiments.

Article Snippet: Western analyses of PVDF membranes utilized established protocols and antibodies for DNMT1 (Abcam #Ab54759), mouse anti-flag (Sigma cat# F3165-.2MG), rabbit anti-PBRM1 (ABCAM cat# ab86156), rabbit anti c-MYC (Cell signaling cat# 5605), rabbit anti-p27/CDKN1B (Cell signaling Ab cat# 3833) and β-actin (Sigma, #a3854).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Control, Western Blot, Staining

Journal: Cell reports

Article Title: PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation

doi: 10.1016/j.celrep.2021.109747

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Western analyses of PVDF membranes utilized established protocols and antibodies for DNMT1 (Abcam #Ab54759), mouse anti-flag (Sigma cat# F3165-.2MG), rabbit anti-PBRM1 (ABCAM cat# ab86156), rabbit anti c-MYC (Cell signaling cat# 5605), rabbit anti-p27/CDKN1B (Cell signaling Ab cat# 3833) and β-actin (Sigma, #a3854).

Techniques: Virus, Recombinant, Protease Inhibitor, cDNA Synthesis, DNA Purification, Software