Journal: PLoS ONE

Article Title: Decreased Camptothecin Sensitivity of the Stem-Cell-Like Fraction of Caco2 Cells Correlates with an Altered Phosphorylation Pattern of Topoisomerase I

doi: 10.1371/journal.pone.0099628

Figure Lengend Snippet: ( A ) The TopI activity in CD44+ cell extracts after phosphorylation or dephosphorylation. Whole cell extract was either left untreated (indicated by Control) or incubated with phosphatase (indicated by Dephos) or CK2 (indicated by Phos) before TopI activity was measured by REEAD in the presence of 60 µM CPT or 5% DMSO as indicated in the figure. One example randomly picked out of 12 individual microscopic images of each reaction sample is shown in the top panel. A quantitative depiction of three independent experiments, obtained as described in the legend of is shown in the lower panel. ( B ) Same as ( A ), except that the analyses were performed on extracts from CD44− cells.

Article Snippet: CD44 MicroBeads (human), FcR Blocking Reagent (human), CD44-PE Antibody (human), CD133-PE Antibody (human), Casein Kinase II (CK2) and alkaline phosphatase from calf intestine were purchased from New England BioLabs Ltd. (UK).

Techniques: Activity Assay, De-Phosphorylation Assay, Incubation

Journal: Cell Genomics

Article Title: NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors

doi: 10.1016/j.xgen.2024.100550

Figure Lengend Snippet: NRDE2 interacts with CK2 complex and facilitates its assembly and activity (A) Two independent IP-MS assays showing the associations of CK2 complex (CK2A1/CK2A2/CK2B) and NRDE2. IP assays were performed in HEK293T cells transiently overexpressing Flag-NRDE2 or empty vector. CK2, casein kinase 2; IP-MS, immunoprecipitation in combination with mass spectrometry; kDa, kilodalton. (B) Co-immunoprecipitation (Co-IP) assays showing the interactions between NRDE2 and the components of CK2 complex (CK2A1/CKA2/CK2B) in HepG2 cells. (C) GST pull-down assays showing the interactions between NRDE2 and CK2A1, CK2A2, and CK2B, respectively. GST-tagged wild-type NRDE2 (GST-N) was expressed and purified from E. coli . His-tagged CK2A1, CK2A2, and CK2B were expressed and purified from HEK293T cells. (D) NRDE2 co-localized with CK2A1/CK2A2/CK2B at nucleus in HepG2 cells with no ionizing radiation (IR) or camptothecin (CPT) treatment by immunofluorescence assays. Fluorescence-intensity profiles were obtained using ImageJ software (v1.8.0), along a straight line (white) crossing the nucleus of a representative cell. (E) The effects of transiently enforced expression of NRDE2-WT or NRDE2-N377I on the assembly of CK2 complex using immunoprecipitation followed by immunoblotting assays in HepG2 cells. (F) LacO/LacR chromatin-targeting protein interaction assays showing the interactions between NRDE2-WT or NRDE2-N377I and CK2A1 in U2OS-lacO cells without IR or CPT treatment. The U2OS-lacO cells were transiently transfected with GFP-CK2A1 and Myc-LacR-NRDE2 (WT or N377I). (G) The interactions between CK2A1 and CK2A2/CK2B assessed by LacO/LacR chromatin-targeting protein interaction assays in U2OS-lacO cells without IR or CPT treatment. The cells were transiently transfected with GFP-LacR-CK2A1, mCherry-CK2B with empty vector, Flag-NRDE2-WT, or Flag-NRDE2-N377I. (H) The effects of endogenous NRDE2 on DNA damage induced recruitments of CK2A1/CK2A2/CK2B to chromatin in HepG2 cells treated with camptothecin (CPT, 10 μM) were investigated by chromatin fractionation assays. Western blotting assays for the total or chromatin fractions from HepG2 cells with stable knockdown of NRDE2 or stably enforced expression of NRDE2 at 4 h after CPT treatment using the indicated antibodies. (I) The effects of knockdown or enforced expression of NRDE2 on cellular CK2 activity in HepG2 cells. We used the phosphorylation levels of CK2 substrates as a readout of cellular CK2 activity. NRDE2 was stably knocked down, and the NRDE2-WT and NRDE2-N377I were transiently expressed in cells. The data are shown as the mean ± standard error of mean (SEM) of three independent experiments. ∗∗∗ p < 0.001 by two-tailed unpaired Student’s t test.

Article Snippet: Protein lysates were then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting assays were performed using the antibodies specific to NRDE2 (1:1,000; 24968-1-AP, Proteintech, China), ATM (1:1,000; 2873T, Cell Signaling Technology, USA), ATR (1:1,000; 2790S, Cell Signaling Technology, USA), CHK1 (1:1,000; 2360S, Cell Signaling Technology, USA), CHK2 (1:1,000; 3440S, Cell Signaling Technology, USA), CK2A1 (1:2,000; 10992-1-AP, Proteintech, China), CK2A2 (1:2,000; 10606-1-AP, Proteintech, China), CK2B (1:2,000; NBP1-06514, Nouvs Biologicals, USA), CK2 phosphorylation (1:1,000; 8738, Cell Signaling Technology, USA), Flag (1:2,000; F3165, Sigma, USA), HA (1:2,000; M180-3, MBL, Japan), Histone H3 (1:1,000; 17168-1-AP, Proteintech, China), MDC1 (1:500; NB100-395, Nouvs Biologicals, USA), MTR4 (1:1,000; 12719-2-AP, Proteintech, China), MYH9 (1:1,000; 11128-1-AP, Proteintech, China), NPM1 (1:1,000; 10306-1-AP, Proteintech, China), RTEL1 (1:1,000; 25337-1-AP, Proteintech, China), pan-p-S/T (1:1,000; AP1067, ABclonal, China), p -ATR (1:1,000; 2853T, Cell Signaling Technology, USA), p -ATM (1:1,000; 5883T, Cell Signaling Technology, USA), p -CHK1 (1:1,000; 2348T, Cell Signaling Technology, USA), p -CHK2 (1:1,000; 2197S, Cell Signaling Technology, USA), RANBP17 (1:1,000; DF4379, Affinity, USA), STEAP3 (1:1,000; 17186-1-AP, Proteintech, China) and β-actin (1:5,000; 60008-1-Ig, Proteintech, China), respectively.

Techniques: Activity Assay, Protein-Protein interactions, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Purification, Immunofluorescence, Fluorescence, Software, Expressing, Western Blot, Transfection, Fractionation, Knockdown, Stable Transfection, Phospho-proteomics, Two Tailed Test

Journal: Cell Genomics

Article Title: NRDE2 deficiency impairs homologous recombination repair and sensitizes hepatocellular carcinoma to PARP inhibitors

doi: 10.1016/j.xgen.2024.100550

Figure Lengend Snippet:

Article Snippet: Protein lysates were then subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blotting assays were performed using the antibodies specific to NRDE2 (1:1,000; 24968-1-AP, Proteintech, China), ATM (1:1,000; 2873T, Cell Signaling Technology, USA), ATR (1:1,000; 2790S, Cell Signaling Technology, USA), CHK1 (1:1,000; 2360S, Cell Signaling Technology, USA), CHK2 (1:1,000; 3440S, Cell Signaling Technology, USA), CK2A1 (1:2,000; 10992-1-AP, Proteintech, China), CK2A2 (1:2,000; 10606-1-AP, Proteintech, China), CK2B (1:2,000; NBP1-06514, Nouvs Biologicals, USA), CK2 phosphorylation (1:1,000; 8738, Cell Signaling Technology, USA), Flag (1:2,000; F3165, Sigma, USA), HA (1:2,000; M180-3, MBL, Japan), Histone H3 (1:1,000; 17168-1-AP, Proteintech, China), MDC1 (1:500; NB100-395, Nouvs Biologicals, USA), MTR4 (1:1,000; 12719-2-AP, Proteintech, China), MYH9 (1:1,000; 11128-1-AP, Proteintech, China), NPM1 (1:1,000; 10306-1-AP, Proteintech, China), RTEL1 (1:1,000; 25337-1-AP, Proteintech, China), pan-p-S/T (1:1,000; AP1067, ABclonal, China), p -ATR (1:1,000; 2853T, Cell Signaling Technology, USA), p -ATM (1:1,000; 5883T, Cell Signaling Technology, USA), p -CHK1 (1:1,000; 2348T, Cell Signaling Technology, USA), p -CHK2 (1:1,000; 2197S, Cell Signaling Technology, USA), RANBP17 (1:1,000; DF4379, Affinity, USA), STEAP3 (1:1,000; 17186-1-AP, Proteintech, China) and β-actin (1:5,000; 60008-1-Ig, Proteintech, China), respectively.

Techniques: Phospho-proteomics, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Cell Fractionation, CCK-8 Assay, Sequencing, RNA Sequencing, Software

Journal: Journal of neurochemistry

Article Title: The polysialic acid mimetics idarubicin and irinotecan stimulate neuronal survival and neurite outgrowth and signal via protein kinase C

doi: 10.1111/jnc.14076

Figure Lengend Snippet: Cell signaling essential for neurite outgrowth that is triggered by idarubicin, irinotecan, epirubicin and CA.Bar diagram displays the average longest neurite length (mean + SEM) of rat cerebellar granule cells pre-treated with different signal transducer molecule inhibitors (KT 5720 (PKA), HBDDE (PKC), TBCA (CKII), Erk inhibitor III (Erk), PP121 (Src), 1-Naphthyl PP1 (Fyn), bpV(HOpic) (PTEN)) and untreated cells or cells treated with 1 nM idarubicin or epirubicin, 0.01 nM irinotecan or 30 μg/ml CA. The hash sign shows a significant difference between the untreated group (control; -) and the groups treated only with PSA mimetics or CA (#p< 0.001). Asterisks signify statistically significant differences within the groups as determined by one-way ANOVA with Fisher's PLSD test (n = 300 cells from 6 wells out of 3 independent experiments; F=36.575 p < 0.0001; PLSD *p< 0.05).

Article Snippet: Protein kinase C inhibitor 2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol-dimethyl ether (HBDDE), casein kinase II inhibitor (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA), Erk inhibitor 1-nitro-2-[(Z)-[5-(3-nitrophenyl)furan-2-yl]methylideneamino]guanidine (Erk inhibitor III), phosphatase and tensin homolog (PTEN) and protein phosphotyrosine phosphatase (PTP) inhibitor dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (bpv(HOpic)) and were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Control

Journal: Journal of neurochemistry

Article Title: The polysialic acid mimetics idarubicin and irinotecan stimulate neuronal survival and neurite outgrowth and signal via protein kinase C

doi: 10.1111/jnc.14076

Figure Lengend Snippet: Cell signaling triggered by idarubicin, irinotecan, epirubicin and CA which is essential for neuronal survival. (a) Inhibitors do not influence basal cell survival. Bar diagram displays the average number of living murine cerebellar granule cells (mean + SEM) treated with different signal transducer molecule inhibitors (KT 5720 (PKA), HBDDE (PKC), TBCA (CKII), ERK inhibitor III (Erk), PP121 (Src), 1-Naphthyl PP1 (1-N-PP1; Fyn), bpV(HOpic) (bpV; PTEN)). (b) Bar diagram displays the average number of living murine cerebellar granule cells (mean + SEM) pre-treated with different signal transducer molecule inhibitors and treated with 1 nM idarubicin, irinotecan or epirubicin or 30 μg/ml CA followed by cell death induction with H2O2. For each treatment and experiment 6 wells were used and the experiment was repeated four times (n = 4). The pound sign shows significant difference between the untreated group (-; - H2O2) and the stressed group (-; + H2O2) or the stressed groups treated with compounds (-; + H2O2; idarubicin/irinotecan/CA) and the stressed groups treated with inhibitors and compounds (inhibitor; + H2O2; idarubicin/irinotecan/CA) (#p< 0.001). Asterisks signify statistically significant difference compared to the hydrogen peroxide treated group as determined by one-way ANOVA with test Holm-Sidak post-hoc test (*p< 0.005).

Article Snippet: Protein kinase C inhibitor 2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol-dimethyl ether (HBDDE), casein kinase II inhibitor (E)-3-(2,3,4,5-tetrabromophenyl)acrylic acid (TBCA), Erk inhibitor 1-nitro-2-[(Z)-[5-(3-nitrophenyl)furan-2-yl]methylideneamino]guanidine (Erk inhibitor III), phosphatase and tensin homolog (PTEN) and protein phosphotyrosine phosphatase (PTP) inhibitor dipotassium bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate (bpv(HOpic)) and were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques:

Journal: Cancer & Metabolism

Article Title: Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l -type amino acid transporter 1

doi: 10.1186/s40170-022-00295-8

Figure Lengend Snippet: Changes in the phosphorylation of substrates of suggested key kinases and their regulatory proteins by LAT1 inhibition. Protein extracted from cells treated with 30 μM JPH203 and control cells was analyzed by Western blot. (A and B) The decrease in phosphorylation associated with JPH203 treatment for indicated time: Thr-389 of p70 S6K ( A ) and Ser-1469 of TOP2A ( B ). The data are presented as a ratio of the signal intensity of phosphorylated protein to total protein ± SD ( n = 3). * p value < 0.05. ns = not significant (one sample t test) ( C ) Decreased phosphorylation of part of CK2 substrates by LAT1 inhibition with JPH203 for 24 h. Phosphorylation on the consensus CK2 substrate motif was detected by a specific antibody. D Decreased phosphorylation of Ser-209 of CK2β, a regulatory subunit of CK2, in cells treated with JPH203 for 24 h. E CK2 activity of BTC cells treated with JPH203. The extracted protein of BTC cells treated with 30 μM JPH203 for 24 h was subject to CK2 activity assay by ELISA using p53 N-terminal peptide and p53-pS46 antibody conjugated with horseradish peroxidase. F The decrease of CK2α co-immunoprecipitated with NOLC1. Protein extracted from KKU-055 and KKU-213 cells treated with JPH203 for 24 h was subject to immunoprecipitation using anti-NOLC1 antibody

Article Snippet: In Western blotting, the following antibodies were used at the indicated dilutions: p70 S6K–pT389 (1:1000, #9234), p70 S6K (1:1000, #9202), phospho–CK2 substrate (1:1000, #8738) from Cell Signaling Technology (Danvers, MA, USA); CK2β (1:500, 22418-1-AP), NOLC1 (1:1000, 11815-1-AP), and β-actin (1:5000, 66009-1-Ig) from Proteintech Group (Chicago, IL, USA); TOP2A-pS1469 (1:1000, PA5-64536) from Affinity Biosciences (Cincinnati, OH, USA); CK2β–pS209 (1:1000, 44-1090G) from Invitrogen (Waltham, MA, USA); TOP2A (1:200, sc-365916), CK2α (1:1000, sc-373894) from Santa Cruz Biotechnology (Dallas, TX, USA); goat anti-mouse IgG-HRP (1:5000, 115-035-062), goat anti-rabbit IgG-HRP (1:5000, 111-035-003) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Techniques: Phospho-proteomics, Inhibition, Control, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Cancer & Metabolism

Article Title: Phosphoproteomics revealed cellular signals immediately responding to disruption of cancer amino acid homeostasis induced by inhibition of l -type amino acid transporter 1

doi: 10.1186/s40170-022-00295-8

Figure Lengend Snippet: Evaluation of the combined treatment of JPH203 and CX-4945. A Effect of the combination of JPH203 with CX-4945 on cell proliferation. BTC cell lines were treated with JPH203 or CX-4945 alone, or in combination. After 3 days of treatment, cell growth was assessed by WST assay. The absorbance at 450 nm of each sample was expressed as % of the control without JPH203 treatment. B Wound healing assay using KKU-100 treated with 30 μM JPH203 or 5μM CX-4945 alone, or in combination. C The number of migrating cells of KKU-055 and KKU-100 treated with 30 μM JPH203 or 5 μM CX-4945 alone, or in combination. The significance of the difference between the treatment with each inhibitor alone and the combination was determined by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent means ± SD ( n = 4 for WST assay, n = 3 for wound healing assay). *Tukey post-hoc p value < 0.05. D Decreased phosphorylation of part of CK2 substrates by LAT1 inhibition with 30 μM JPH203 and 1 μM CX-4945, or in combination for 24 h. Phosphorylation on the consensus CK2 substrate motif was detected by a specific antibody

Article Snippet: In Western blotting, the following antibodies were used at the indicated dilutions: p70 S6K–pT389 (1:1000, #9234), p70 S6K (1:1000, #9202), phospho–CK2 substrate (1:1000, #8738) from Cell Signaling Technology (Danvers, MA, USA); CK2β (1:500, 22418-1-AP), NOLC1 (1:1000, 11815-1-AP), and β-actin (1:5000, 66009-1-Ig) from Proteintech Group (Chicago, IL, USA); TOP2A-pS1469 (1:1000, PA5-64536) from Affinity Biosciences (Cincinnati, OH, USA); CK2β–pS209 (1:1000, 44-1090G) from Invitrogen (Waltham, MA, USA); TOP2A (1:200, sc-365916), CK2α (1:1000, sc-373894) from Santa Cruz Biotechnology (Dallas, TX, USA); goat anti-mouse IgG-HRP (1:5000, 115-035-062), goat anti-rabbit IgG-HRP (1:5000, 111-035-003) from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Techniques: WST Assay, Control, Wound Healing Assay, Comparison, Phospho-proteomics, Inhibition