human mk2  (Carna Inc)

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Cascade assay for target engagement confirms the mechanisms of action for reference compounds. (A) Schematic of MK2/3 signaling. (B) Isolated reactions tested in the biochemical cascade assays. (C) Concentration-response profiles for CC-99677 and ATI-450 against active kinases on their own (open symbols) or for cascade assays using active p38α to activate unactive MK2 or MK3 (solid symbols). Phosphorylation of the respective substrates for the individual enzymes, or in the cascade assays for the unactive enzyme (MK2 or MK3), were measured by HTRF.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Drug discovery, Isolation, Concentration Assay, Phospho-proteomics

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Dual HTRF assay development. (A) Schematic of the dual HTRF assay targeting free MK2 bound to a covalent biotinylated tracer (biotin|cov; see panel (C)). (B) Determination of specific antibody pairs for detecting total MK2. Competition (left) of unlabeled monoclonal antibodies for the epitope of anti-MK2 D1E11 terbium (Tb)-cryptate, enabling the identification of a minimally competitive antibody for quantification of total MK2. Unlabeled antibodies were titrated into reactions containing 0.5× anti-MK2 D1E11 Tb-cryptate and 10 nM biotin|cov tracer paired with streptavidin Alexa Fluor 488. Mouse anti-MK2 7H4.2 was chosen for further assay development and directly conjugated to Alexa Fluor 633 for enhanced HTRF signal. Specificity of this antibody pair for MK2 was tested against recombinant human MK2 or MK3 (right). The antibody pair anti-MK3 D54E4 Tb-cryptate with anti-MK3 2B5 Alexa Fluor 568 was used as a positive control for detection of MK3. Note the cross-reactivity of anti-D1E11 Tb-cryptate for MK3. (C) Structure of biotin|cov, the tracer used for the dual HTRF assays (left). The specificity of this tracer for recombinant human MK2 over MK3 is shown in the associated plot (right), using 0.5× anti-MK2 D1E11-Tb cryptate as the energy donor. (D) CC-99677 target engagement on 10 nM recombinant human MK2 using the dual HTRF assay reagents (anti-MK2 pair plus biotin|cov with streptavidin Alexa Fluor 488). The cause of the observed incomplete target occupancy by this compound is unknown. (E) Quantification of endogenous MK2 protein abundance in human cell lines (left) and murine RAW264.7 cells or splenocytes (right). Specificity of the chosen anti-MK2 pair was demonstrated by the absence of signal from genetic knockout samples (human U937 and mouse splenocytes). HCC1428 possessed the highest detectable concentration of MK2 among the adherent human cell lines (family of red symbols), so it was used for studies to measure endogenous target engagement.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: HTRF Assay, Bioprocessing, Assay Development, Recombinant, Positive Control, Drug discovery, Quantitative Proteomics, Knock-Out, Concentration Assay

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: NanoBRET assay development. (A) Emission spectrum of NanoLuciferase measured live in dimethyl sulfoxide-treated HeLa cells over-expressing either amino- or carboxy-terminal MK2 fusions, with or without 0.17 mM (0.004%) Triton X-100 permeabilization (dashed or solid lines, respectively). The filter band passes used to measure NanoBRET signal are highlighted (centered at 452 nm for NanoLuciferase and 600 nm for BODIPY585). Magnified portion of the spectrum (right) shows the fraction of light from the NanoLuciferase donor that overlaps with –and should be subtracted from– the BODIPY acceptor emission signal. (B) Characterization of BODIPY|rev and BODIPY585|cov tracers, which compete for the same binding pocket as CC-99677. Structures of tracers (left) are shown alongside magnified emission spectra when added to HeLa cells transfected with plasmids encoding either amino- or carboxy-terminal MK2 fusions (middle) as well as the ratiometric NanoBRET signal following a titration of each tracer (right). Signal was measured 1 hour after tracer addition to the live intact (solid) or permeabilized (open/dashed line) cells using a CLARIOstar Plus (average of 5 replicates with standard deviation is shown). Line weight and style follow the legend in panel (A). (C) and (D) NanoBRET signal kinetics in the presence of BODIPY585|cov tracer, using well-by-well acquisitions (C) on the CLARIOstar Plus (CLARIOstar) or full-plate imaging (D) with a GNF Systems luminescence plate reader (LPR). NanoLuciferase signal (blue profiles in shaded background) decayed over time whereas the NanoBRET ratio (red profiles in white background) increased with exposure time to tracer. Continuous measurements for longer than 1 hour were achieved by supplementing with Endurazine substrate. (E) and (F) Measurement of k -on (E) and k -off (F) for CC-99677 using NanoBRET with BODIPY585|cov on a luminescence plate reader. The time points denote when CC-99677-containing medium was exchanged for medium containing excess BODIPY585|cov, which irreversibly quenched the system by saturating free binding sites. The profile for a reversible compound that occupies the same pocket as the covalent CC-99677 (gray, open circles) is included in the k -off plots (F).

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Assay Development, Expressing, Binding Assay, Transfection, Titration, Standard Deviation, Imaging, Microplate Reader Luminescence Measurement

Journal: RSC Chemical Biology

Article Title: High-throughput assay for measuring target occupancy of covalent compounds: a case study with MK2

doi: 10.1039/d5cb00224a

Figure Lengend Snippet: Comparison of compound target occupancy assays. (A) to (C) Measurement of MK2 target occupancy using dual HTRF, for endogenous protein targeting, in human HCC1428 cells (A) or murine RAW264.7 cells (B) versus NanoBRET in HeLa cells transfected with NanoLuciferase amino-terminally fused to human MK2 (C). Cells were treated with compound for 3 hours before washout. Curve fits are shown for the “free” signal (the ratio of “tracer”/“total”) for CC-99677 treatment, with regression ( R 2 ) values of 0.96 (RAW264.7 in HTRF); 0.92 (HCC1428 in HTRF), and 0.99 (HeLa in NanoBRET). (D) and (E) NanoBRET target occupancy data for 3-hour CC-99677 exposure in the presence of increasing concentrations of fetal bovine or human serum (D) to model in vivo compound availability using the NanoBRET assay. The right-shifted phenotype of CC-99677 is independent of human serum lot (E), suggesting that the free-fraction of CC-99677 compound will be reduced in circulation.

Article Snippet: Recombinant murine MK2 (TP506027) was purchased from OriGene (Rockville, MD, U.S.A.) whereas active human MK2 (02-142) and MK3 (02-143) were purchased from Carna Biosciences (Natick, MA, U.S.A.).

Techniques: Comparison, Transfection, In Vivo

Journal: The Journal of Biological Chemistry

Article Title: Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway

doi: 10.1016/j.jbc.2023.104699

Figure Lengend Snippet: MK2 catalyzes RSK1 phosphorylation at Ser-380. A , HeLa cells were treated with 10 μM MK2 inhibitor III (MK2 inh III) for 30 min and then stimulated with 10 ng/ml EGF for 10 min or 50 μM anisomycin for 20 min. B , HeLa cells were transfected with siRNA against MK2 or the negative control. At 48 h post-transfection, cells were stimulated with 10 ng/ml EGF for 10 min or 50 μM anisomycin for 20 min. A and B , Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, phospho-MK2 (pMK2), and α-Tubulin. Red arrow , phosphorylated MK2; blue arrow , non-phosphorylated MK2. C , HEK293 cells were transfected with expression vectors for EGFP-tagged kinase-dead EphA2 (EphA2-KD-EGFP), FLAG-tagged RSK1, FLAG-tagged p38α, MK2 (wild-type (WT) or kinase-dead mutant (KD)), and/or an empty vector. At 24 h post-transfection, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, FLAG, MK2, and β-Actin. D , HeLa cells were treated with 10 μM MK2 inhibitor III and then stimulated with 100 μM CDDP for 3 h or 0.3 M NaCl (Osmo) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, pMK2, and α-Tubulin. E , immunoprecipitated RSK1 and RSK2 prepared from HeLa cells were incubated with recombinant human active GST-MK2 at 30 °C for 30 min. Reaction mixtures were analyzed by immunoblotting with anti-phospho-RSK (Ser-380 of RSK1; Ser-386 of RSK2), RSK1, RSK2, and pMK2 antibodies. EphA2, ephrin type-A receptor 2; MK2, MAPK-activated protein kinase 2, RSK, p90 ribosomal S6 kinase.

Article Snippet: Recombinant human EGF was obtained from R&D Systems; recombinant human active GST-MK2 protein was from Carna Biosciences; a phos-tag ligand, anisomycin, NaCl, and CDDP were from Wako Pure Chemical Industries; SB203580 and TMZ were from Merck KGaA; MK2 inhibitor III and GSK2334470 were from MedChemExpress; BI-D1870 was from BioVision; BIRB796, Skepinone-L, and BX-702 were from Selleck Chemicals.

Techniques: Transfection, Negative Control, Expressing, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Incubation, Recombinant, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway

doi: 10.1016/j.jbc.2023.104699

Figure Lengend Snippet: MK2 induced the atypical activation of RSK. A and B , HeLa cells were stimulated with 10 ng/ml EGF for 10 min or 50 μM anisomycin for 20 min. C and D , HeLa cells were treated with 10 μM MK2 inhibitor III for 30 min and then stimulated with 10 ng/ml EGF for 10 min ( C ) or 50 μM anisomycin for 20 min ( D ). Whole-cell lysates were separated by Zn 2+ Phos-tag SDS-PAGE, followed by immunoblotting with an anti-phospho-RSK (Ser-380 and Thr-573), RSK1, or RSK2 antibody. The images of RSK1 in A ( dark ) and B ( right ) are from the same blot.

Article Snippet: Recombinant human EGF was obtained from R&D Systems; recombinant human active GST-MK2 protein was from Carna Biosciences; a phos-tag ligand, anisomycin, NaCl, and CDDP were from Wako Pure Chemical Industries; SB203580 and TMZ were from Merck KGaA; MK2 inhibitor III and GSK2334470 were from MedChemExpress; BI-D1870 was from BioVision; BIRB796, Skepinone-L, and BX-702 were from Selleck Chemicals.

Techniques: Activation Assay, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway

doi: 10.1016/j.jbc.2023.104699

Figure Lengend Snippet: MK2-induced EphA2 phosphorylation is independent of the CTK activity of RSK. A and B , HEK293 cells were transfected with the expression vectors for FLAG-tagged RSK1 (wild-type (WT) or CTK-dead mutant (CTKm)), FLAG-tagged constitutively activated MEK1 (MEK1-CA-FLAG), MK2, FLAG-tagged constitutively activated p38α (p38α-CA-FLAG), and/or an empty vector. At 24 h post-transfection, whole-cell lysates were immunoblotted with primary antibodies against phospho-RSK at Ser-380 (pRSK), FLAG, pMK2, MK2, and β-Actin. C – E , HEK293 cells were transfected with the expression vectors for EphA2-KD-EGFP, RSK1-CTKm-FLAG (Ser-380 WT, Ala-substitute mutation (SA) or Ser-221 SA), MK2, p38α-CA-FLAG, and/or an empty vector. At 24 h post-transfection, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, FLAG, pMK2, and β-Actin. F , HeLa cells were treated with 10 μM GSK2334470 for 30 min and then stimulated with 10 ng/ml EGF for 10 min or 50 μM anisomycin for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and α-Tubulin. G , a schematic diagram of RSK phosphorylation induced by MK2 or ERK. CTK, carboxyl-terminal kinase; EphA2, ephrin type-A receptor 2; MK2, MAPK-activated protein kinase 2; NTK, amino-terminal kinase, RSK, p90 ribosomal S6 kinase.

Article Snippet: Recombinant human EGF was obtained from R&D Systems; recombinant human active GST-MK2 protein was from Carna Biosciences; a phos-tag ligand, anisomycin, NaCl, and CDDP were from Wako Pure Chemical Industries; SB203580 and TMZ were from Merck KGaA; MK2 inhibitor III and GSK2334470 were from MedChemExpress; BI-D1870 was from BioVision; BIRB796, Skepinone-L, and BX-702 were from Selleck Chemicals.

Techniques: Activity Assay, Transfection, Expressing, Mutagenesis, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway

doi: 10.1016/j.jbc.2023.104699

Figure Lengend Snippet: The p38-MK2-RSK-EphA2 pathway regulates cell migration. HEK293 cells were transfected with the expression vectors for EphA2-KD-EGFP (Ser-897 WT or SA), RSK1-CTKm-FLAG, p38α-CA-FLAG, MK2, and/or an empty vector. At 24 h post-transfection, cells were treated with DMSO or 10 μM BI-D1870 for 2 h ( C and D ). Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, FLAG, pMK2, and β-Actin ( A and C ). Cell migration was observed using a time-lapse imaging system for 120 min ( B and D ). The accumulated distance of cell migration (μm) was calculated and shown in box and whisker plots. ∗ p < 0.05 by the Tukey–Kramer HSD test. EphA2, ephrin type-A receptor 2; MK2, MAPK-activated protein kinase 2; RSK, p90 ribosomal S6 kinase.

Article Snippet: Recombinant human EGF was obtained from R&D Systems; recombinant human active GST-MK2 protein was from Carna Biosciences; a phos-tag ligand, anisomycin, NaCl, and CDDP were from Wako Pure Chemical Industries; SB203580 and TMZ were from Merck KGaA; MK2 inhibitor III and GSK2334470 were from MedChemExpress; BI-D1870 was from BioVision; BIRB796, Skepinone-L, and BX-702 were from Selleck Chemicals.

Techniques: Migration, Transfection, Expressing, Plasmid Preparation, Imaging, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway

doi: 10.1016/j.jbc.2023.104699

Figure Lengend Snippet: The p38-MK2-RSK-EphA2 pathway promotes U87-MG cell migration induced by TMZ. A , U87-MG cells were treated with DMSO or 100 μM temozolomide (TMZ) for 48 h. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pp38, p38, pHSP27, HSP27 and β-Actin. B , U87-MG cells were transfected with siRNA against EphA2 (#1 or #2) or the negative control. After 5 h of transfection, cells were treated with DMSO or 100 μM TMZ for 72 h. Cell migration was observed using a time-lapse imaging system for 120 min. The accumulated distance of cell migration (μm) was calculated and shown in box and whisker plots. ∗ p < 0.05 by the Tukey–Kramer HSD test. Whole-cell lysates were immunoblotted with primary antibodies against EphA2 and β-Actin. C – F , U87-MG cells were treated with DMSO or 100 μM TMZ for 72 h, then treated with DMSO, 10 μM BI-D1870 ( C and E ) or 10 μM MK2 inhibitor III ( D and F ) for 2 h. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pHSP27, HSP27, and β-Actin ( C and D ). Cell migration was observed using a time-lapse imaging system for 120 min and the accumulated distance of cell migration (μm) was calculated and shown in box and whisker plots ( E and F ). ∗ p < 0.05 by the Tukey–Kramer HSD test. DMSO, dimethyl sulfoxide; EphA2, ephrin type-A receptor 2; HSP27, heat shock protein 27; RSK, p90 ribosomal S6 kinase.

Article Snippet: Recombinant human EGF was obtained from R&D Systems; recombinant human active GST-MK2 protein was from Carna Biosciences; a phos-tag ligand, anisomycin, NaCl, and CDDP were from Wako Pure Chemical Industries; SB203580 and TMZ were from Merck KGaA; MK2 inhibitor III and GSK2334470 were from MedChemExpress; BI-D1870 was from BioVision; BIRB796, Skepinone-L, and BX-702 were from Selleck Chemicals.

Techniques: Migration, Transfection, Negative Control, Imaging, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway

doi: 10.1016/j.jbc.2023.104699

Figure Lengend Snippet: Model of the non-canonical activation of EphA2 by typically or atypically activated RSK. Upon the stimulation of growth factors, ERK typically activates CTK to induce the activation of NTK, resulting in the non-canonical activation of EphA2. On the other hand, cellular stress-activated MK2 catalyzes the phosphorylation of RSK1 at Ser-380 to induce NTK activation in a CTK-independent manner. This atypically activated RSK also induces the non-canonical activation of EphA2 to promote cancer malignancy. CTK, carboxyl-terminal kinase; EphA2, ephrin type-A receptor 2; ERK, extracellular signal-regulated kinase; MK2, MAPK-activated protein kinase 2; NTK, amino-terminal kinase; RSK, p90 ribosomal S6 kinase.

Article Snippet: Recombinant human EGF was obtained from R&D Systems; recombinant human active GST-MK2 protein was from Carna Biosciences; a phos-tag ligand, anisomycin, NaCl, and CDDP were from Wako Pure Chemical Industries; SB203580 and TMZ were from Merck KGaA; MK2 inhibitor III and GSK2334470 were from MedChemExpress; BI-D1870 was from BioVision; BIRB796, Skepinone-L, and BX-702 were from Selleck Chemicals.

Techniques: Activation Assay

Journal: Cell Death & Disease

Article Title: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma

doi: 10.1038/s41419-020-2563-4

Figure Lengend Snippet: a Left: Coomassie brilliant blue stained SDS-PAGE gel separating immunoprecipitated subcellular fractions of cells with exogenous OCT4 expression using anti-FLAG antibody. M: Marker, C: cytosolic, N: nuclear. For protein ID, the entire lanes were analyzed, and for PTM, the specific OCT4 band was subjected to mass spectrometry analyses. Right: OCT4 protein sequence with PTM status identified by mass spectrometry. Underline: mass spectrometry coverage, purple: not phosphorylated or acetylated, Red: phosphorylated, acetylated, or undetermined. b Predicted PTM sites in NTD and POUs domains of OCT4 interacting with/phosphorylated by MK2 (S 111 ) and DNA-PKcs (S 93 ). c Left: Direct nuclear interaction of MK2 or DNA-PKcs with OCT4 confirmed by immunoprecipitation in LHN-R cells stably transduced with the doxycycline-inducible construct of wild-type OCT4 with mycDDK-tag. Right: The direct interaction between OCT4 and MK2 is also confirmed by Ni-NTA pull down (top panel), and also by immunoprecipitation by FLAG using purified proteins (bottom panel). d Effect of stable MAPKAPK2 knockdown on OCT4 and c-MYC protein expression in LHN-R (selected for resistance to 13- cis RA in the laboratory) and COG-N-508h (established from a PD patient sample after 13- cis RA treatment). The results were reproducible in a repeat experiment. e Protein expression of OCT4, MK2, Cyclin A, and NeuN (mature neuronal marker) in LHN-R cells with MAPKAPK2 knockdown. Cells stably transduced with non-targeting NT-shRNA or MAPKAPK2 -shRNA were treated with vehicle control or 5 μM 13- cis RA for 14 days. The results were reproducible in a repeat experiment. f Reversal of 13- cis RA resistance shown as neurite outgrowth in MAPKAPK2 knockdown LHN-R cells. Cells stably transduced with non-targeting NT-shRNA or MAPKAPK2 -shRNA were treated with vehicle control or 5 μM 13- cis RA for 14 days. A scale bar: 100 μM. g Reversal of 13- cis RA resistance shown as cell cycle arrest in MAPKAPK2 knockdown LHN-R cells. Cells stably transduced with non-targeting NT-shRNA (S-phase cells: 11 ± 0.5% vs 9 ± 2.2%, p = 0.11) or MAPKAPK2 -shRNA (S-phase cells: 8.5 ± 0.1% vs 3.2 ± 0.2%, p < 0.01) were treated with vehicle control or 13- cis RA for 14 days. Left: representative histograms, right: percentage of cells in each phase of cell cycle.

Article Snippet: Chloroquine diphosphate (CQ), cycloheximide (CHX), isotretinoin (13- cis RA), NaF, NaHCO 3 , Na 3 VO 4 , Tris-HCl, Triton X-100, Aprotinin, Leupeptin, Pepstatin A, PMSF, Ethanol (molecular biology), Isopropanol (molecular biology), ITS, Puromycin, and 3× FLAG peptide were from Sigma-Aldrich; DTT, EDTA, Formaldehyde (molecular biology), Glycine (molecular biology), IPTG, NaCl, MES, SDS, TAE, Tween-20, FBS, DMEM, IMDM, RPMI-1640, l -Glutamine, Pen Strep, Sodium pyruvate, Trypsin/EDTA, Lipofectamine®, PLUS TM reagent, Proteinase K, RNase A, and Superscript® III First-Strand Synthesis System from ThermoFisher Scientific; Tet-free FBS and Doxycycline from Clontech; Ni-NTA from EMD Millipore; NH 2 -terminal His-tagged human recombinant OCT4 protein (purity > 90%, made in E. coli ) from ProteinONE; COOH-terminal mycDDK-tagged human recombinant MK2 protein from OriGene; MK2 inhibitor PF3644022 from Tocris; MK2 inhibitor III from Santa Cruz Biotechnoloyg; bortezomib from LC Laboratories; p38 inhibitor SB203580 from Sellectchem; Age1-HF , BamH1-HF , EcoR1-HF , Mlu1-HF , Not1-HF , Pme1 , Sgf1 , Xba1 , and Xho1 restriction enzymes from New England Biolabs; bovine serum albumin from Jackson ImmunoResearch Laboratories; All oligonucleotides were synthesized from Integrated DNA Technologies (IDT).

Techniques: Staining, SDS Page, Immunoprecipitation, Expressing, Marker, Mass Spectrometry, Sequencing, Stable Transfection, Transduction, Construct, Purification, Knockdown, shRNA, Control

Journal: Cell Death & Disease

Article Title: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma

doi: 10.1038/s41419-020-2563-4

Figure Lengend Snippet: a Positive correlation between mRNA expression of MYC and MAPKAPK2 in 249 neuroblastoma patients. Expression data from the neuroblastoma NCI TARGET database ( https://ocg.cancer.gov/programs/target/data-matrix ). b Inverse correlation between MYCN and MAPKAPK2 mRNA expression in 249 neuroblastoma patients (Data: NCI TARGET database https://ocg.cancer.gov/programs/target/data-matrix ). c Overall survival of patients by MAPKAPK2 mRNA expression in neuroblastoma from the NCI TARGET database. Of the total patients ( n = 247), only patients with MYCN non-amplification ( n = 175) were used for the analysis. The data was scanned to identify maximum separation of the curves, and the p -value was adjusted by Bonferroni adjustment. The overall survival using the median MAPKAPK2 expression and the event-free survival are shown in Supplementary Fig. . d MK2 protein expression by immunohistochemistry staining in neuroblastoma primary tumors collected at diagnosis with low expression of both MYCN and c-MYC proteins (upper panel) and with high c-MYC protein (lower panel) expression. A white scale bar: 20 μM, a black scale bar: 20 μM. e Constructs (plasmid: pGEX-4T-1) encoding a human wild-type OCT4 or OCT4 mutants (S93A and S111A) with a GST tag and thrombin cleavage site at the NH 2 -terminus. f Human recombinant OCT4 proteins expressed in BL21/DE3 strain of E. coli after IPTG induction, GST column purification, and thrombin cleavage subjected to SDS-PAGE and stained with Coomassie brilliant blue solution. g Proteins in f detected by immunoblotting using anti-OCT4 antibody. h In vitro kinase assay of MK2 on phosphorylating OCT4WT and OCT4 S111A . The results were reproducible in a repeat experiment.

Article Snippet: Chloroquine diphosphate (CQ), cycloheximide (CHX), isotretinoin (13- cis RA), NaF, NaHCO 3 , Na 3 VO 4 , Tris-HCl, Triton X-100, Aprotinin, Leupeptin, Pepstatin A, PMSF, Ethanol (molecular biology), Isopropanol (molecular biology), ITS, Puromycin, and 3× FLAG peptide were from Sigma-Aldrich; DTT, EDTA, Formaldehyde (molecular biology), Glycine (molecular biology), IPTG, NaCl, MES, SDS, TAE, Tween-20, FBS, DMEM, IMDM, RPMI-1640, l -Glutamine, Pen Strep, Sodium pyruvate, Trypsin/EDTA, Lipofectamine®, PLUS TM reagent, Proteinase K, RNase A, and Superscript® III First-Strand Synthesis System from ThermoFisher Scientific; Tet-free FBS and Doxycycline from Clontech; Ni-NTA from EMD Millipore; NH 2 -terminal His-tagged human recombinant OCT4 protein (purity > 90%, made in E. coli ) from ProteinONE; COOH-terminal mycDDK-tagged human recombinant MK2 protein from OriGene; MK2 inhibitor PF3644022 from Tocris; MK2 inhibitor III from Santa Cruz Biotechnoloyg; bortezomib from LC Laboratories; p38 inhibitor SB203580 from Sellectchem; Age1-HF , BamH1-HF , EcoR1-HF , Mlu1-HF , Not1-HF , Pme1 , Sgf1 , Xba1 , and Xho1 restriction enzymes from New England Biolabs; bovine serum albumin from Jackson ImmunoResearch Laboratories; All oligonucleotides were synthesized from Integrated DNA Technologies (IDT).

Techniques: Expressing, Amplification, Immunohistochemistry, Staining, Biomarker Discovery, Construct, Plasmid Preparation, Recombinant, Purification, SDS Page, Western Blot, In Vitro, Kinase Assay

Journal: Cell Death & Disease

Article Title: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma

doi: 10.1038/s41419-020-2563-4

Figure Lengend Snippet: a Specificity of anti-pOCT4S111-antibody tested in HHN-R cells with exogenous expression of OCT4WT and OCT4 S111A , and OCT4 S93A . b Specificity of anti-pOCT4S111-antibody tested by neutralizing antibody with pOCT4 S111 peptide or OCT4 S111 peptide, and detecting pOCT4 S111 in LHN-R cells, stably expressing OCT4WT. c c-MYC, OCT4, phosphoOCT4 (pOCT4 S111 ), MK2, and phosphoMK2 (pMK2 T334 ) expression in patient-derived neuroblastoma cell lines established from samples obtained pre-therapy at diagnosis from patients with high-risk neuroblastoma. CHLA-20: control for two different membranes. c-MYC and GAPDH protein levels are from Fig. . d c-MYC, OCT4, phosphoOCT4 (pOCT4 S111 ), MK2, and phosphoMK2 (pMK2 T334 ) expression in patient-derived neuroblastoma cell lines established from progressive disease clinical samples. CHLA-20: control for two different membranes. * Patient not treated with 13- cis RA prior to obtaining sample. c-MYC and GAPDH protein levels are from Fig. . e Dot plots quantitating immunoblotting data from a and b . The values were normalized in two ways by the expression of specific proteins in CHLA-20 and GAPDH. f Dot plots of c-MYC and pMK2 protein expression comparing cell lines established at Dx to cell lines established at PD from patients treated with 13- cis RA ( cis RA), and at PD from patients not treated with 13- cis RA (no cis RA) cell lines. ** and *: significant, ns: not significant. g c-MYC, OCT4, phosphoOCT4 (pOCT4 S111 ), MK2, and phosphoMK2 (pMK2 T334 ) expression in matched pairs of cell lines established at diagnosis (Dx) and progressive disease (PD) from seven different neuroblastoma patients. CHLA-20 was used as the control for each membrane. CHLA-78 (Dx) and CHLA-95 (PD) is a pair established from patients before 13- cis RA became standard of care for high-risk neuroblastoma. * Patient not treated with 13- cis RA prior to obtaining sample.

Article Snippet: Chloroquine diphosphate (CQ), cycloheximide (CHX), isotretinoin (13- cis RA), NaF, NaHCO 3 , Na 3 VO 4 , Tris-HCl, Triton X-100, Aprotinin, Leupeptin, Pepstatin A, PMSF, Ethanol (molecular biology), Isopropanol (molecular biology), ITS, Puromycin, and 3× FLAG peptide were from Sigma-Aldrich; DTT, EDTA, Formaldehyde (molecular biology), Glycine (molecular biology), IPTG, NaCl, MES, SDS, TAE, Tween-20, FBS, DMEM, IMDM, RPMI-1640, l -Glutamine, Pen Strep, Sodium pyruvate, Trypsin/EDTA, Lipofectamine®, PLUS TM reagent, Proteinase K, RNase A, and Superscript® III First-Strand Synthesis System from ThermoFisher Scientific; Tet-free FBS and Doxycycline from Clontech; Ni-NTA from EMD Millipore; NH 2 -terminal His-tagged human recombinant OCT4 protein (purity > 90%, made in E. coli ) from ProteinONE; COOH-terminal mycDDK-tagged human recombinant MK2 protein from OriGene; MK2 inhibitor PF3644022 from Tocris; MK2 inhibitor III from Santa Cruz Biotechnoloyg; bortezomib from LC Laboratories; p38 inhibitor SB203580 from Sellectchem; Age1-HF , BamH1-HF , EcoR1-HF , Mlu1-HF , Not1-HF , Pme1 , Sgf1 , Xba1 , and Xho1 restriction enzymes from New England Biolabs; bovine serum albumin from Jackson ImmunoResearch Laboratories; All oligonucleotides were synthesized from Integrated DNA Technologies (IDT).

Techniques: Expressing, Stable Transfection, Derivative Assay, Biomarker Discovery, Control, Western Blot, Membrane

Journal: Cell Death & Disease

Article Title: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma

doi: 10.1038/s41419-020-2563-4

Figure Lengend Snippet: a OCT4 at S111 residue is phosphorylated in LHN-R. OCT4WT and OCT4 S111A mutant were exogenously expressed in LHN-R cells. The expression of pOCT4 S111 , OCT4, c-MYC, and Cyclin A was evaluated in subcellular fractions. b Assessment of DNA-binding ability of wild-type OCT4 and mutant OCT4 S111A using MYC −1209/−1140 / MYC reporter assay system. Empty vector, POU5F1-mycDDK and POU5F1 S111A -mycDDK (4 μg each) were separately co-transfected with reporter gene MYC −1209/−1140 /DDK-MYC-mER TM (4 μg) in HEK293FT cells. After 48 h, the equal amount of protein lysates (20 μg) were run and analyzed by SDS/PAGE and WB using specific antibodies, as indicated. Anti-ERα: DDK-c-MYC-mER TM expression, anti-DDK (FLAG): expression of exogenous mycDDK-tagged wild-type OCT4 and its mutant. c OCT4 stability was decreased in OCT4 S111A mutant relatively to OCT4 WT , shown by cycloheximide (CHX) treatment. The cells were transduced with a doxycycline-inducible system to exogenously express OCT4 WT or OCT4 S111A mutant, treated with doxycycline (Dox) for 48 h, followed by cycloheximide (CHX) incubation for various times before immunoblotting. d Rescue of OCT4 S111A protein degradation by bortezomib. The LHN-R cells were transduced with a Dox-inducible system to exogenously express OCT4 S111A mutant, treated with Dox for 24 h, followed by 10 μg/ml of cycloheximide (CHX) along with Bortezomib (1 μM, proteasome inhibitor) or Chloroquine (50 μM, lysosome inhibitor) incubation for 6 h before immunoblotting. DMSO was served as a vehicle control. e Decreased pOCT4 S111 , OCT4, c-MYC, and Cyclin A in CHLA-20 (PD cell line from patients not treated with 13- cis RA), LHN-R, COG-N-289, COG-N-334, and COG-N-415 (the last three PD cell lines were established from patients treated with 13- cis RA) treated with an MK2 inhibitor (PF3644022). f Decreased pOCT4 S111 , OCT4, c-MYC, and Cyclin A in COG-N-289 and COG-N-415 (the PD cell lines established from patients treated with 13- cis RA) treated with an MK2 inhibitor (MK2iIII). g Relative viability of neuroblastoma cells (two cell lines with high c-MYC: COG-N-469h and COG-N-508h, two cell lines with low c-MYC: COG-N-322 and COG-N-503h) treated with PF3644022 (0–300 nM) for 96 h in six replicates. h Relative viability of neuroblastoma cells (two sets of matched pairs: COG-N-442h and COG-N-443h, COG-N-532h, and COG-N-547h) treated with PF3644022 (0–10 μM) for 96 h in six replicates. The results were reproducible in a repeat experiment.

Article Snippet: Chloroquine diphosphate (CQ), cycloheximide (CHX), isotretinoin (13- cis RA), NaF, NaHCO 3 , Na 3 VO 4 , Tris-HCl, Triton X-100, Aprotinin, Leupeptin, Pepstatin A, PMSF, Ethanol (molecular biology), Isopropanol (molecular biology), ITS, Puromycin, and 3× FLAG peptide were from Sigma-Aldrich; DTT, EDTA, Formaldehyde (molecular biology), Glycine (molecular biology), IPTG, NaCl, MES, SDS, TAE, Tween-20, FBS, DMEM, IMDM, RPMI-1640, l -Glutamine, Pen Strep, Sodium pyruvate, Trypsin/EDTA, Lipofectamine®, PLUS TM reagent, Proteinase K, RNase A, and Superscript® III First-Strand Synthesis System from ThermoFisher Scientific; Tet-free FBS and Doxycycline from Clontech; Ni-NTA from EMD Millipore; NH 2 -terminal His-tagged human recombinant OCT4 protein (purity > 90%, made in E. coli ) from ProteinONE; COOH-terminal mycDDK-tagged human recombinant MK2 protein from OriGene; MK2 inhibitor PF3644022 from Tocris; MK2 inhibitor III from Santa Cruz Biotechnoloyg; bortezomib from LC Laboratories; p38 inhibitor SB203580 from Sellectchem; Age1-HF , BamH1-HF , EcoR1-HF , Mlu1-HF , Not1-HF , Pme1 , Sgf1 , Xba1 , and Xho1 restriction enzymes from New England Biolabs; bovine serum albumin from Jackson ImmunoResearch Laboratories; All oligonucleotides were synthesized from Integrated DNA Technologies (IDT).

Techniques: Residue, Mutagenesis, Expressing, Binding Assay, Reporter Assay, Plasmid Preparation, Transfection, SDS Page, Transduction, Incubation, Western Blot, Control

Journal: Cell Death & Disease

Article Title: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma

doi: 10.1038/s41419-020-2563-4

Figure Lengend Snippet: a c-MYC, OCT4, phosphoOCT4 (pOCT4 S111 ), MK2, and phosphoMK2 (pMK2 T334 ) expression in patient-derived xenografts (PDXs) of neuroblastoma established from clinical samples of obtained at diagnosis ( n = 8) and at progressive disease ( n = 9). CHLA-20 was used as the control for the two membranes. * COG-N-603x and COG-N-623x are matched Dx-PD pair of PDXs established from the same patients. The matched-pair direct-to-culture cell lines are COG-N-603h and COG-N-623h shown in Fig. . b Dot plots quantitating immunoblotting data from a . The values were normalized in two ways by the expression of specific proteins in CHLA-20 and GAPDH. c Correlation between c-MYC and pOCT4 S111 , c-MYC and pMK T334 , and pOCT4 S111 and pMK T334 . The results of linear regression analyses with 95% confidence interval (dotted line) are presented ( n = 17). d Proposed mechanism of c-MYC transcriptional activation in progressive disease neuroblastoma. MK2 shuttles into the nuclei of cells and phosphorylates OCT4 at S111 residue. There are two OCT4-binding sites in the c-MYC promoter/enhancer region between −1209 and −1140. The binding of pOCT4 S111 increases transcriptional activation of MYC .

Article Snippet: Chloroquine diphosphate (CQ), cycloheximide (CHX), isotretinoin (13- cis RA), NaF, NaHCO 3 , Na 3 VO 4 , Tris-HCl, Triton X-100, Aprotinin, Leupeptin, Pepstatin A, PMSF, Ethanol (molecular biology), Isopropanol (molecular biology), ITS, Puromycin, and 3× FLAG peptide were from Sigma-Aldrich; DTT, EDTA, Formaldehyde (molecular biology), Glycine (molecular biology), IPTG, NaCl, MES, SDS, TAE, Tween-20, FBS, DMEM, IMDM, RPMI-1640, l -Glutamine, Pen Strep, Sodium pyruvate, Trypsin/EDTA, Lipofectamine®, PLUS TM reagent, Proteinase K, RNase A, and Superscript® III First-Strand Synthesis System from ThermoFisher Scientific; Tet-free FBS and Doxycycline from Clontech; Ni-NTA from EMD Millipore; NH 2 -terminal His-tagged human recombinant OCT4 protein (purity > 90%, made in E. coli ) from ProteinONE; COOH-terminal mycDDK-tagged human recombinant MK2 protein from OriGene; MK2 inhibitor PF3644022 from Tocris; MK2 inhibitor III from Santa Cruz Biotechnoloyg; bortezomib from LC Laboratories; p38 inhibitor SB203580 from Sellectchem; Age1-HF , BamH1-HF , EcoR1-HF , Mlu1-HF , Not1-HF , Pme1 , Sgf1 , Xba1 , and Xho1 restriction enzymes from New England Biolabs; bovine serum albumin from Jackson ImmunoResearch Laboratories; All oligonucleotides were synthesized from Integrated DNA Technologies (IDT).

Techniques: Expressing, Derivative Assay, Biomarker Discovery, Control, Western Blot, Activation Assay, Residue, Binding Assay