Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Expression and roles of p16 INK4a , p21 Cip1 and p27 Kip1 in the response of lung adenocarcinoma cell line models to long-term treatment with Dex. H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days and whole cell lysates were probed by western blot for p16 INK4a , p21 Cip1 and p27 Kip1 with GAPDH as the loading control (Panel A). H1299GR Clone 4 cells were transduced with either non-targeted control shRNA or P21 Cip1 shRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were extracted and probed by western blot for p21 Cip1 , p27 Kip1 and GAPDH (loading control) (Panel B). In parallel, cells treated as described for Panel B, were plated for colony formation in 6-well plates in triplicates without further treatment (Panel C). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and treated with Dex (100 nM) for the indicated number of days, when the cells were harvested and whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel D). In parallel, cells transfected as described for Panel D, and treated with Dex (100 nM) were plated on days 0 and 5 to measure colony formation in 6-well plates in triplicates without further treatment (Panel E) and also harvested on days 0 and 3 of treatment for measuring cell size (Panel F). H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or p27 Kip1 siRNA and 24 h later they were treated with Dex (100 nM) or vehicle for 3 days and the cells were stained to assess expression of β- galactosidase (blue staining) (Panel G, left); in parallel, the treated cells were lysed and analyzed by western blot for p27 expression (Panel G, right). H1299GR Clone 4 cells were transduced with either non-targeted control vector or pLenti-p27 expression vector and after the indicated number of days, whole cell lysates were probed by western blot for p27 Kip1 or GAPDH (loading control) (Panel H). In parallel, cells were harvested for measuring the cell size (Panel I) and also plated to measure colony formation in 6-well plates in triplicate (Panel J). Panel C: *P, 0.0004; **P, 0.0006; § P, 0.0008; ‡ P, 0.00015. Panel E: *P, 0.016. Panel J: *P, 0.015; § P, 0.00006; ‡ P, 0.00007.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Western Blot, Control, Transduction, shRNA, Transfection, Staining, Plasmid Preparation

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Mechanism of accumulation of p27 Kip1 in response to long-term Dex treatment in high GR expressing cells. H1299GR Clone 4 cells were transfected with either non-targeted control siRNA or a combination of FOXO3 and FOXO4 siRNAs and treated with Dex (100 nM) for the indicated number of days, when whole cell lysates were probed by western blot for FOXO3, FOXO4, p27 Kip1 and GAPDH (loading control) (Panel A). H292, A549, H1299, H1299GR Clone 2 and H1299GR Clone 4 cells were treated with Dex (100 nM) for the indicated number of days, when RNA was extracted and relative expression levels of p27 Kip1 mRNA were measured using real time RT-PCR (Panel B). H1299GR Clone 4 cells were pre-treated with vehicle or Dex (100 nM) for 72 h. The cells were then treated with 1ug/ml of actinomycin D and at the time points indicated, RNA was harvested from the cells and p27 Kip1 mRNA was measured by real time RT-PCR to determine p27 Kip1 mRNA turnover rates. The mRNA values are plotted relative to the values at the time of addition of actinomycin D (Panel C). Panel B (H292): *P, 0.01; § P, 0.008. Panel B (A549): *P, 0.02; § P, 0.0001. Panel B (H1299GRα clone2): *P, 0.0001; ‡ P, 0.0007, § P, 0.001. Panel B (H1299GRα clone4): *P, 0.0001; ‡ P, 0.001; § P, 0.0001.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

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

Journal: Scientific Reports

Article Title: Chronic p27 Kip1 Induction by Dexamethasone Causes Senescence Phenotype and Permanent Cell Cycle Blockade in Lung Adenocarcinoma Cells Over-expressing Glucocorticoid Receptor

doi: 10.1038/s41598-018-34475-8

Figure Lengend Snippet: Effect of Dex on mouse tumor xenografts expressing low or high levels of GR. SCID mice bearing xenografts of parental H1299 cells (Panel A) or H1299GR Clone 4 cells (Panel B) were implanted with either placebo or Dex (2.5 mg) slow-release pellets at the time indicated by the arrow. The tumor growth was monitored via caliper measurements and tumor volumes were calculated using the formula noted in the Methods section. At the time of sacrifice, residual H1299GR Clone 4 tumors were harvested from the placebo (Panel C, left) and Dex (Panel C, right) treated mice and stained to ensure GR expression and to quantify p27 Kip1 by immunohistochemistry (Panel D). The scoring criteria based on staining intensity and percent positivity, as outlined in the Methods section. The parental H1299 cell tumor xenografts harvested at the end of placebo or Dex treatments in Panel A were also examined by immunohistochemistry for GR and p27 Kip1 expression (Panel E). Panel B: *P, 0.08; § P, 0.016; ‡ P, 0.0015.

Article Snippet: Blasticidin and puromycin were from Thermo Fisher Scientific and Sigma-Aldrich, St. Louis, MO, respectively. p21 Cip1 shRNA was from Sigma-Aldrich and FOXO3 (#L-003007-00-0005), FOXO4 (#L-003016-00-0005) and p27 Kip1 siRNAs (#L-003472-00-0005) were from GE Dharmacon (Lafayette, CO). pLenti-p27 Kip1 expression vector (#RC201661L1) was purchased from Origene, Rockville, MD.

Techniques: Expressing, Staining, Immunohistochemistry

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: Nature Communications

Article Title: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

doi: 10.1038/s41467-017-01742-7

Figure Lengend Snippet: GJA4 regulates endothelial cell cycle arrest via CDKN1B. a CDKN1B mRNA expression was significantly reduced in Gja4 −/− P6 retinal endothelial cells (CD31+/CD45−) (mean relative mRNA expression ± SEM vs. Gja4 +/− littermate controls; n = 3 ( Cdk4 , Gja4 −/− ; Cdk6 , Gja4 −/− Ccne1 , Gja4 +/− ; Ccne2 , Gja4 +/− ), n = 4 ( Cdk4 , Gja4 +/− ; Cdk6 , Gja4 +/− ; Ccnb1 ; Cdkn1a , Gja4 −/− ), n = 5 ( Ccne1 , Gja4 −/− ), n = 6 ( Ccne2 , Gja4 −/− ; Cdkn1a , Gja4 +/− ; Ccnd1 , Gja4 −/− ), n = 8 ( Cdk2 ; Tp53 , Gja4 −/− ), n = 9 ( Ccnd1 , Gja4 +/− ; Cdkn1b ; Tp53 , Gja4 −/− ); individual values plotted where n < 5). b CDKN1B expression was detected in Gja4 +/+ , but not Gja4 −/− , endothelial cells of remodeling retinal vessels. (Colors: PECAM1 (red), CDKN1B (green); scale bar: 50 µm). c CDKN1B protein was elevated in HUVEC by 16 h FSS, and this effect was abolished by si- GJA4 (mean densitometry ± SEM vs. Static; n = 3 for all groups; one-way ANOVA: p = 0.002, asterisks indicate p < 0.05 in post hoc t -test). Uncropped western blots presented in Supplementary Fig. . d Constitutive GJA4 expression (lenti- Gja4 ) arrested endothelial cells in G1 and reduced actively cycling cells in S/G2/M, while si- CDKN1B had the opposite effect, regardless of GJA4 expression (mean difference in cell cycle % ± SEM vs. Control; n = 6; one-way ANOVA: p = 0.0002 (G1), p = 0.0003 (S/G2/M), asterisks indicate p < 0.05 in post hoc t -test vs. si-Ctrl + lenti-Ctrl). e GJA4 knockdown (si- GJA4 ) reduced the proportion of endothelial cells in G1 and increased the proportion in S/G2/M, and constitutive expression of CDKN1B (lenti- CDKN1B ) had the opposite effect, regardless of GJA4 expression (mean difference in cell cycle % ± SEM vs. Control; n = 3 for all groups; one-way ANOVA: p = 0.002 (G0), p < 0.0001 (G1), p < 0.0001 (S/G2/M), asterisks indicate p < 0.05 in post hoc t -tests vs. si-Ctrl+ lenti-Ctrl). f CDKN1B phosphorylation at serine 10 (pCDKN1B (S10)) and total protein levels were reduced by si- GJA4 and rescued with lenti- Gja4 . Uncropped blots presented in Supplementary Fig. . g MAPK/ERK signaling inhibition by 1 h treatment with 20 µM U0126, an inhibitor of MEK1/2, blocked GJA4-dependent CDKN1B phosphorylation at serine 10 and reduced total CDKN1B protein levels. Uncropped blots presented in Supplementary Fig.

Article Snippet: For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si- GJA4 (Dharmacon L-011669-00), si- CDKN1B (Dharmacon L-040178-00), si- NOTCH1 (Dharmacon L-007771-00), si- NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti- Gja4 ) , human CDKN1B (lenti- CDKN1B , cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma).

Techniques: Expressing, Western Blot, Control, Knockdown, Phospho-proteomics, Inhibition

Journal: Nature Communications

Article Title: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

doi: 10.1038/s41467-017-01742-7

Figure Lengend Snippet: Endothelial cell cycle arrest, per se, enables arterial gene expression. a Treatment of HUVEC with 10 µM clotrimazole or 2 µM palbociclib reduced RB1, phosphorylated RB1 (pRb1), and E2F1, suggestive of G1 arrest. Clotrimazole tended to upregulate CDKN1B expression and preserve GJA4 expression, and CDK4 expression was lost only with CDK4/6i treatment. Uncropped blots presented in Supplementary Fig. . b Using FACS to assess cell cycle distribution, clotrimazole was found to arrest HUVEC in G1, even when CDKN1B was knocked down (via si- CDKN1B ) (mean difference in cell cycle % ± SEM vs. DMSO; n = 3 (Clotrimazole), n = 5 (DMSO), n = 6 (Clotrimazole+ si- CDKN1B ); individual values plotted where n < 5; one-way ANOVA: p = 0.007 (G1), p = 0.03 (S/G2/M); asterisks indicate p < 0.05 in post hoc t -tests). c Clotrimazole upregulated EFNB2 and GJA5 regardless of CDKN1B expression (mean relative mRNA expression ± SEM vs. DMSO; n = 4 (Clotrimazole, p27), n = 5 (Clotrimazole, EFNB2 ; Clotrimazole, GJA5 ), n = 7 (Clotrimazole+ si-CDKN1B), n = 8 (DMSO); individual values plotted where n < 5; one-way ANOVA: p = 0.03 ( EFNB2 , GJA5 ), p = 0.0007 ( CDKN1B )). d Palbociclib treatment also arrested endothelial cells in G1 (mean difference in cell cycle % ± SEM vs. DMSO; n = 3 for all groups; Students’ t -test: p = 0.002 (G1), p < 0.0001 (S/G2/M)), e upregulated EFNB2 and GJA5 (Cx40) mRNA levels, abolished CDK4 expression, and had no effect on GJA4 (Cx37) and CDKN1B (p27), (mean relative mRNA expression ± SEM vs DMSO; n = 9 ( GJA4 ), n = 12 ( EFNB2 ), n = 14 ( GJA5 ), n = 16 ( CDKN1B ; CDK4 ); Students’ t -test: p = 0.02 ( EFNB2 , GJA5 ), p < 0.0001 ( CDK4 )). f Endothelial cells within arteries and surrounding plexi of P6 retinas from Cdt1 -mOrange+ reporter mice were predominantly in G1 phase, whereas endothelial cells in adjacent veins were not. (Colors: IB4 (red), ERG (blue), pH3 (magenta), CDT1 (green); scale bar: 100 µm). g We hypothesize that in remodeling vessels, arterial shear activates a novel Notch-Cx37-p27 signaling pathway that promotes endothelial cell cycle arrest to enable arterial gene expression

Article Snippet: For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si- GJA4 (Dharmacon L-011669-00), si- CDKN1B (Dharmacon L-040178-00), si- NOTCH1 (Dharmacon L-007771-00), si- NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti- Gja4 ) , human CDKN1B (lenti- CDKN1B , cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma).

Techniques: Gene Expression, Expressing, Shear

Journal: Nature Communications

Article Title: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification

doi: 10.1038/s41467-017-01742-7

Figure Lengend Snippet: FSS-NOTCH-GJA4-CDKN1B axis regulates arterial identity genes. a Disorganized or absent SMA investment was observed in Gja4 −/− and WT + DAPT mice, compared to WT controls. (Colors: PECAM1 (red), SMA (white); scale bar: 100 µm; symbols: a = artery, v = vein, R = remodeling, M = mature). b Radial SMA investment of arteries, as well as c radial distance of GJA5 (Cx40) expression, were reduced in Gja4 −/− and DAPT-treated animals (mean radial distance as % of total vascular outgrowth ± SEM vs WT; n = 3 (WT + DAPT; WT, GJA5), n = 4 (WT, SMA), n = 6 ( Gja4 −/− ); one-way ANOVA: p = 0.03 (SMA), p = 0.04 (GJA5); asterisks indicate p < 0.05 in post hoc t -tests vs. WT). d DLL4 activation of NOTCH signaling upregulated EFNB2 (EphrinB2) and GJA5 , and this effect was abolished with si- GJA4 or si- CDKN1B (mean relative mRNA expression ± SEM vs. PBS; n = 3 (si- GJA4 ; si- CDKN1B, EFNB2 ), n = 4 (si-Ctrl, GJA5 ; si- CDKN1B , GJA5 ), n = 4 (si-Ctrl, EFNB2 ); Students’ t -test: p = 0.02 (si-Ctrl, EFNB2 ), p = 0.04 (si-Ctrl, GJA5 ), p = 0.0005 (si- GJA4 , GJA5 ), p < 0.0001 (si- CDKN1B , GJA5 )). e NOTCH activation was maximal with 1 h exposure to 18 dynes/cm 2 shear (mean NICD R.F.U. ± SEM; n = 7000 cells, representative of N = 3). f Expression of GJA4 (Cx37), GJA5 (Cx40) and EFNB2 (EphrinB2) were also maximal at 18 dynes/cm 2 (mean relative mRNA expression ± SE; n = 3, representative of 4 experiments). g Knockdown of NOTCH1, GJA4 or CDKN1B reduced basal and 10 h FSS-induced upregulation of EFNB2 and GJA5 (mean relative mRNA expression ± SEM vs. Static; n = 3 technical replicates; representative of two biological replicates)

Article Snippet: For siRNA knockdown, cells pre-conditioned with EGM-2 (Lonza) for >16 h were transfected with 20 nM si- GJA4 (Dharmacon L-011669-00), si- CDKN1B (Dharmacon L-040178-00), si- NOTCH1 (Dharmacon L-007771-00), si- NOTCH4 (Dharmacon L-011883-00) or control siRNA (Ambion AM4611) for >96 h. For lentiviral transduction, HUVEC were pre-infected for 48 h with lentivirus containing mouse Gja4 (lenti- Gja4 ) , human CDKN1B (lenti- CDKN1B , cloned from Addgene 14049) or empty vector in presence of 10 μg/mL polybrene (Sigma).

Techniques: Expressing, Activation Assay, Shear, Knockdown

Journal: OncoTargets and Therapy

Article Title:

Hypermethylation Of ADHFE1 Promotes The Proliferation Of Colorectal Cancer Cell Via Modulating Cell Cycle Progression

doi: 10.2147/ott.s223423

Figure Lengend Snippet: Figure 3 ADHFE1 modulates cell cycle progression. (A) GSEA analyses of the “REACTOME_CELL_CYCLE” and “REACTOME_G1_S_TRANSITION” gene sets in the low versus high expression group of ADHFE1 in CRC. (B and C) Flow-cytometry analyses of the cell cycle progression in the indicated CRC cells. Error bars represent the means ± SD from three independent experiments. *p<0.05, **p<0.01. (D) Western blot analyses of p53, p21, p27 and CyclinD1 in the indicated cells. GAPDH was used as a loading control.

Article Snippet: The PVDF membrane was subsequently blocked in PBST solution containing 5% non-fat milk and incubated at 4°C overnight with specific antibodies anti-ADHFE1 (1:1000 dilution; GeneTex Inc, Texas, St. Antonio, USA), anti-CyclinD1 (1:500 dilution; ProteinTech Group, Chicago, IL, USA), anti-p21 (1:500 dilution; ProteinTech Group, Chicago, IL, USA), anti-p27 (1:1000 dilution; ProteinTech Group, Chicago, IL, USA), anti-p53 (1:2000 dilution; ProteinTech Group, Chicago, IL, USA) and anti-GAPDH (1:5000 dilution; ProteinTech Group, Chicago, IL, USA).

Techniques: Expressing, Flow Cytometry, Western Blot, Control