Journal: Molecular Cancer Research

Article Title: Inhibition of Expression of the Chromatin Remodeling Gene, SNF2L, Selectively Leads to DNA Damage, Growth Inhibition, and Cancer Cell Death

doi: 10.1158/1541-7786.mcr-09-0119

Figure Lengend Snippet: FIGURE 4. Knockdown of SNF2L caused DNA damage and resulted in a DNA damage response in the HM lines. A. MDA-MB-468 cells were transfected with SNF2L siRNA or NCSI. Forty-eight hours after transfection, DNA damage was analyzed by the Comet assay and the results showed damaged DNA (the comet tail) outside the nucleus after treatment of SNF2L siRNA (bottom) compared with undamaged DNA in the cells treated with NCSI (top). B. The per- centage of cells with damaged DNA is depicted in representative HM, LG, and NU lines. There was a marked increase in DNA damage in the HM lines. C. The MDA-MB-468 cells that had been so treated were also analyzed for the levels of both total and phosphorylated proteins involved in a DNA damage response, including ATR, BRCA1, CHK1, CHK2, and H2AX. Increased phosphorylation of all these proteins was observed with SNF2L knockdown but no alterations of the respective total proteins.

Article Snippet: Antibodies used for detection of DNA damage were obtained from the DNA Damage Antibody Sampler (Cell Signaling Technology, Inc.), which included Phospho-ATR (Ser428), Phospho-ATM (Ser1981) mouse monoclonal antibody (mAb), Phospho-BRCA1 (Ser1524), Phospho-CHK1 (Ser296), Phospho-CHK2 (Thr68), PhosphoHistone H2AX (Ser139), and Phospho-p53 (Ser15; 16G8) mouse mAb. p53 (7F5) rabbit mAb and β-actin (13E5) rabbit mAb were also used (Cell Signaling Technology, Inc.).

Techniques: Knockdown, Transfection, Single Cell Gel Electrophoresis, Phospho-proteomics

Journal: Molecular Cancer Research

Article Title: Inhibition of Expression of the Chromatin Remodeling Gene, SNF2L, Selectively Leads to DNA Damage, Growth Inhibition, and Cancer Cell Death

doi: 10.1158/1541-7786.mcr-09-0119

Figure Lengend Snippet: FIGURE 7. DNA damage, DNA damage response, and apoptosis. A. Flow cytometry of cells stained in Annexin-V and PI solution and treated with NCSI (left), SNF2L siRNA (center), and SNF2L siRNA with a general caspase inhibitor (right). Only with SNF2L knockdown alone was there an increase in both early (Annexin-V) and late (PI) apoptosis (top right). Inhibition of apoptosis abolished this effect (bottom) and the control negative siRNA (NCSI) produced no increased apoptosis (top left). B. Comet assay confirmed that DNA damage was induced by SNF2L knockdown (center) and was not observed when the negative control (NCSI) was used (left). Significantly, DNA damage still occurred even when apoptosis was inhibited with a general caspase inhibitor (right). C. Western blot indicated that DNA damage and the resultant DNA damage response, e.g., p-H2AX from SNF2L knockdown preceded and did not follow apoptosis. SNF2L siRNA, but not NCSI, increased cleaved PARP (a surrogate of apoptosis) and p-H2AX. Specific caspase inhibitors to caspase 3, caspase 8, and caspase 9 were only minimally effective at inhibiting cleaved PARP. The general caspase inhibitor I, on the other hand, showed significant inhibition of PARP cleavage (apoptosis), yet p-H2AX was still expressed.

Article Snippet: Antibodies used for detection of DNA damage were obtained from the DNA Damage Antibody Sampler (Cell Signaling Technology, Inc.), which included Phospho-ATR (Ser428), Phospho-ATM (Ser1981) mouse monoclonal antibody (mAb), Phospho-BRCA1 (Ser1524), Phospho-CHK1 (Ser296), Phospho-CHK2 (Thr68), PhosphoHistone H2AX (Ser139), and Phospho-p53 (Ser15; 16G8) mouse mAb. p53 (7F5) rabbit mAb and β-actin (13E5) rabbit mAb were also used (Cell Signaling Technology, Inc.).

Techniques: Flow Cytometry, Staining, Knockdown, Inhibition, Control, Produced, Single Cell Gel Electrophoresis, Negative Control, Western Blot

Journal: Molecular human reproduction

Article Title: Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

doi: 10.1093/molehr/gaw065

Figure Lengend Snippet: Figure 2 Effect of S1P on the expression of endothelial adherens junction proteins in endothelial cells in the presence of FF from controls and patients at risk of OHSS. Densitometric quantification of (A) phospho-vascular endothelial (p-VE)-cadherin, (B) VE-cadherin, (C) N-cadherin and (D) β-catenin. Representative immunoblots are shown in the lower panel. Data are expressed as means ± SEM normalized to β-actin of three independent experiments using 20 control patients and 20 OHSS patients at risk of OHSS (*P < 0.05).

Article Snippet: The blot was preincubated in blocking buffer (5% nonfat milk, 0.05% Tween 20 in 20 mM TBS pH 8.0) for 1 h at room temperature and incubated overnight in blocking buffer at 4°C with appropriate primary antibodies: β-actin 1/3000 (sc-1616-R), N-cadherin 1/200 (sc-7939), vascular endothelial (VE)-cadherin 1/100 (sc-9989) and β-catenin 1/200 (sc-59737) (Santa Cruz Biotechnology, Inc., Santa Cruz, USA), phospho-VE-cadherin 1/1000 (MAB1002) (Millipore, MA, USA), VEGF 1/1000 (ab46154) (Abcam, Cambridge, USA), KDR (VEGFR-2) 1/1000 (D5B1) (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: iScience

Article Title: IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer

doi: 10.1016/j.isci.2024.108984

Figure Lengend Snippet: EGFR/DNAPKcs-mediated DSB NHEJ signaling upregulated in Olaparib-resistant prostate cancer (A) The protein expression of phospho-EGF receptor (Tyr1068) and IGFBP3 in parental cells, OlapR cells was determined by western blot analysis. Tubulin served as a loading control. (B) The protein expression of phospho-DNA-PKcs (Ser2056) and IGFBP3 in parental cells, OlapR cells was determined by western blot analysis. Tubulin served as a loading control. (C) The cell growth of 2B-OlapR cells transfected with EGFR siRNA for 3 days and 5 days was determined by CCK-8. (D) In the cBioPortal database (SUC2/PCF Dream Team, PNAS 2019), EGFR gene expression levels were determined in tumor samples of different Gleason scores. (E) In the cBioPortal database (Broad/Cornell, Nat Genetics 2012), PRKDC (DNA-PKcs) gene expression levels were determined in tumor samples of different Gleason scores. (F) In the cBioPortal database (MSK, Science 2022), PRKDC (DNA-PKcs) gene expression levels were determined in organoid derived cancer types, prostate adenocarcinoma and prostate. p value less than 0.05 was considered significant (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001). Statistical analysis was performed using Student’s t test.

Article Snippet: Phospho-DNA-PKcs (Ser2056) , Cell Signaling Technology , Cat#: 68716, RRID: AB_2939025.

Techniques: Expressing, Western Blot, Control, Transfection, CCK-8 Assay, Gene Expression, Derivative Assay

Journal: iScience

Article Title: IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer

doi: 10.1016/j.isci.2024.108984

Figure Lengend Snippet: IGFBP3 and EGFR are overexpressed in the intrinsic Olaparib-resistant Rv1 cell line (A) The cell growth of Rv1 cells versus LNCaP and C4-2B cells treated with increasing doses of Olaparib for 72 h demonstrate robust resistance to Olaparib was determined by CCK-8. (B) Relative mRNA expression levels of IGFBP3 in Rv1 cells were determined by qPCR. (C) The protein expression of IGFBP3, Phospho-EGF receptor (Tyr1068), and γH2AX in Rv1 cells was determined by western blot analysis. Tubulin served as a loading control. (D) The protein expression of phospho-DNA-PKcs (Ser2056) in Rv1 cells was determined by western blot analysis. Tubulin served as a loading control. (E) The IC50 value of Rv1 cells treated with Gefitinib via cell growth were determined by CCK-8. (F) The cell growth of Rv1 treated with 10 μM Olaparib and 10 μM Gefitinib for 72 h was determined by CCK-8. The combination drug index (CDI) of Olaparib and Gefitinib in Rv1 was calculated. p value less than 0.05 was considered significant (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001). Statistical analysis was performed using Student’s t test.

Article Snippet: Phospho-DNA-PKcs (Ser2056) , Cell Signaling Technology , Cat#: 68716, RRID: AB_2939025.

Techniques: CCK-8 Assay, Expressing, Western Blot, Control

Journal: iScience

Article Title: IGFBP3 promotes resistance to Olaparib via modulating EGFR signaling in advanced prostate cancer

doi: 10.1016/j.isci.2024.108984

Figure Lengend Snippet:

Article Snippet: Phospho-DNA-PKcs (Ser2056) , Cell Signaling Technology , Cat#: 68716, RRID: AB_2939025.

Techniques: Recombinant, Negative Control, Transfection, Reverse Transcription, Protease Inhibitor, Western Blot, Saline, CCK-8 Assay, Viability Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Software

Journal: Journal of Biological Chemistry

Article Title: Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

doi: 10.1074/jbc.m110.201137

Figure Lengend Snippet: FIGURE 1. Inhibition of CK2 decreases the VEGF mRNA decaying activity of TTP. A, CK2 inhibitor DRB decreases the mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR and pcDNA6/V5-TTP. At 24 h post-transfection, cells were treated with CK2 inhib- itor DRB or p38 MAPK inhibitor SB203580 at indicated concentrations for 12 h (A),andluciferase(Luc.)activitywasdetermined.Renillaluciferaseactivitywas normalized to firefly activity. The luciferase activity obtained from psiCHECK2-VEGF 3UTR-transfected cells was set to 1. Each bar represents the mean S.D. of three independent experiments (*, p 0.05; **, p 0.01; ***, p 0.001). ns, not significant. B and C, inhibition of CK2 by siRNA (siCK2) decreases the mRNA decaying activity of TTP. Colo320 cells were cotrans- fected with psiCHECK2-VEGF 3UTR, pcDNA6/V5-TTP, and siCK2 or scRNA. At 24 h post-transfection, expression level of CK2 was determined by immu- noblotting (IB) (B) and luciferase activity was determined (C). Renilla luciferase activitywasnormalizedtofireflyactivity.Theluciferaseactivityobtainedfrom psiCHECK2-VEGF 3UTR-transfected cells was set to 1. Each bar represents the mean S.D. of three independent experiments (**, p 0.01; ***, p 0.001). D, TTP-mediated down-regulation of endogenous VEGF mRNA is attenuated bysiCK2.Colo320cellswerecotransfectedwithpcDNA6/V5-TTPandsiCK2 or scRNA. At 24 h post-transfection, VEGF mRNA was determined by quanti- tative real time PCR. The expression level obtained from mock-transfected cells was set to 1. Each bar represents the mean S.D. of three independent experiments (**,p 0.01; ***, p 0.001).

Article Snippet: SDS-PAGE Analysis and Immunoblotting—Proteins were resolved using SDS-PAGE, transferred onto Hybond-P membranes (GE Healthcare), and probed with appropriate dilutions of antibodies as follows: rabbit anti-human TTP antibody (ab36558, Abcam); anti-human CK2 antibody (sc-12738, Santa Cruz Biotechnology); anti-p38 MAPK antibody (9212, 21578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 24 • JUNE 17, 2011 at Y ork U niversity - O C U L on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from Cell SignalingTechnology,Danvers); anti-phospho-p38MAPK antibody (9215, Cell Signaling); anti-MK2 antibody (3042, Cell Signaling); anti-phospho-MK2 antibody (sc-101729, Santa Cruz Biotechnology); anti-MKP-1 antibody (sc-370, Santa Cruz Biotechnology); anti-phospho-MKP-1 antibody (2857, Cell Signaling Technology); anti-ubiquitin antibody (sc-9133, Santa Cruz Biotechnology); anti-V5 antibody (Genentech, San Francisco); anti-HA antibody (H9658, Sigma); and anti-FLAG antibody (F1804, Sigma).

Techniques: Inhibition, Activity Assay, Transfection, Luciferase, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of Biological Chemistry

Article Title: Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

doi: 10.1074/jbc.m110.201137

Figure Lengend Snippet: FIGURE 6. Effects of CK2 on TTP is mediated by the MKP-1/p38 MAPK pathways. A, CK2 inhibition by DRB increases phosphorylation of p38 MAPK and MK2. Colo320 cells were transfected with pcDNA6/V5-TTP, and the cells were treated with 30 M DRB at 24 h post-transfection. Cells were harvested at indicated times after DRB treatment, and cell lysates were analyzed for TTP, p38 MAPK, phosphorylated p38 MAPK, MK2, and phosphorylated MK2 by immunoblotting (IB). B, mutation of MK2 phosphorylation sites of TTP blocks the DRB-induced degradation of TTP. Colo320 cells were transfected with pcDNA6/V5-TTP or pcDNA6/V5-TTP(S60A and S186A) and were treated with DRB for 12 h. The expression level of the TTP protein was determined by immunoblotting with anti-V5 antibody. C, p38 MAPK inhibitor SB203580 attenuates the decrease mRNA decaying activity of TTP induced by siRNA against CK2. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR, pcDNA6/V5-TTP, and siCK2 or scRNA. Cells were treated with SB203580 for 12 h, and luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from cells transfected with psiCHECK2-VEGF 3UTR was set to 1. Each bar represents the mean S.D. of three independent experiments (***, p 0.001). D, inhibition of CK2 by DRB decreases the expression of MKP-1. Colo320 cells were treated with DRB, and cells were harvested at the indicated times after DRB treatment. Cell lysates were analyzed for MKP-1 by immunoblotting with anti-MKP-1 antibody. E, overexpression of CK2 increases the expression of MKP-1. Colo320 cells were cotransfected with pcDNA6/V5-TTP and pcDNA3/HA-CK2. At 24 h post-transfection, expression of CK2 and MKP-1 was determined by immunoblotting. F, mutation of CK2 phosphorylation sites of MKP-1 blocks the CK2-induced phosphorylation of MKP-1. Colo320 cells were cotransfected with pcDNA6/V5-TTP, pcDNA3/HA-CK2, and pcDNA3.1/FLAG-MKP-1 or pcDNA3.1/FLAG-MKP-1 (S131A and S235A). At 24 h post-transfection, cell lysates were analyzed for CK2, MKP-1, and phosphorylated MKP-1 by immunoblotting. G, inhibition of MKP-1 by siRNA abolishes the effects of CK2 on TTP expression and p38 MAPK phosphorylation. Colo320 cells were cotransfected with pcDNA6/V5-TTP, pcDNA3/HA-CK2, and siRNA against MKP-1 (siMKP-1) or scRNA. At 24 h post- transfection, cell lysates were analyzed for CK2, TTP, MKP-1, p38 MAPK, and phosphorylated p38 MAPK by immunoblotting. H, inhibition of MKP-1 by MKP-1 inhibitor triptolide or siRNA abolishes the effects of CK2 on mRNA decaying activity of TTP. Colo320 cells were cotransfected with psiCHECK2-VEGF 3UTR, pcDNA6/V5-TTP, pcDNA3/HA-CK2, and siMKP-1 or scRNA. Cells were treated with triptolide for 12 h, and the luciferase activity was determined. Renilla luciferase activity was normalized to firefly activity. The luciferase activity obtained from cells cotransfected with psiCHECK2-VEGF 3UTR and pcDNA6/V5-TTP was set to 1. Each bar represents the mean S.D. of three independent experiments (*, p 0.05; ***, p 0.001). ns, not significant.

Article Snippet: SDS-PAGE Analysis and Immunoblotting—Proteins were resolved using SDS-PAGE, transferred onto Hybond-P membranes (GE Healthcare), and probed with appropriate dilutions of antibodies as follows: rabbit anti-human TTP antibody (ab36558, Abcam); anti-human CK2 antibody (sc-12738, Santa Cruz Biotechnology); anti-p38 MAPK antibody (9212, 21578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 24 • JUNE 17, 2011 at Y ork U niversity - O C U L on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from Cell SignalingTechnology,Danvers); anti-phospho-p38MAPK antibody (9215, Cell Signaling); anti-MK2 antibody (3042, Cell Signaling); anti-phospho-MK2 antibody (sc-101729, Santa Cruz Biotechnology); anti-MKP-1 antibody (sc-370, Santa Cruz Biotechnology); anti-phospho-MKP-1 antibody (2857, Cell Signaling Technology); anti-ubiquitin antibody (sc-9133, Santa Cruz Biotechnology); anti-V5 antibody (Genentech, San Francisco); anti-HA antibody (H9658, Sigma); and anti-FLAG antibody (F1804, Sigma).

Techniques: Inhibition, Phospho-proteomics, Transfection, Western Blot, Mutagenesis, Expressing, Activity Assay, Luciferase, Over Expression

Journal: Journal of Biological Chemistry

Article Title: Casein Kinase 2 Regulates the mRNA-destabilizing Activity of Tristetraprolin

doi: 10.1074/jbc.m110.201137

Figure Lengend Snippet: FIGURE 8. Model of regulation of TTP expression by CK2. The p38 MAPK/ MK2 signaling pathway can induce phosphorylation (P) of TTP, which leads to proteasomal degradation of TTP protein. However, when CK2 is activated by TGF- stimulation, it can activate MKP-1, which in turn inhibits the p38 MAPK/ MK2 pathway by dephosphorylation of p38 MAPK, thereby protecting the TTP protein from proteasomal degradation.

Article Snippet: SDS-PAGE Analysis and Immunoblotting—Proteins were resolved using SDS-PAGE, transferred onto Hybond-P membranes (GE Healthcare), and probed with appropriate dilutions of antibodies as follows: rabbit anti-human TTP antibody (ab36558, Abcam); anti-human CK2 antibody (sc-12738, Santa Cruz Biotechnology); anti-p38 MAPK antibody (9212, 21578 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 24 • JUNE 17, 2011 at Y ork U niversity - O C U L on M arch 4, 2015 http://w w w .jbc.org/ D ow nloaded from Cell SignalingTechnology,Danvers); anti-phospho-p38MAPK antibody (9215, Cell Signaling); anti-MK2 antibody (3042, Cell Signaling); anti-phospho-MK2 antibody (sc-101729, Santa Cruz Biotechnology); anti-MKP-1 antibody (sc-370, Santa Cruz Biotechnology); anti-phospho-MKP-1 antibody (2857, Cell Signaling Technology); anti-ubiquitin antibody (sc-9133, Santa Cruz Biotechnology); anti-V5 antibody (Genentech, San Francisco); anti-HA antibody (H9658, Sigma); and anti-FLAG antibody (F1804, Sigma).

Techniques: Expressing, Phospho-proteomics, De-Phosphorylation Assay