mk2 inhibitor pf3644022  (Tocris)

 
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    Tocris mk2 inhibitor pf3644022
    Mk2 Inhibitor Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pf3644022  (Tocris)

     
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    Tocris pf3644022
    Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pf3644022  (Tocris)

     
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    Tocris pf3644022
    Inhibition of p38α or MK2 leaves the G 2 -checkpoint intact in irradiated 82-6 hTert and A549 cells . ( A ) Western blot analysis showing the effect of increasing concentrations of LY2228820, an inhibitor of p38α (p38αi), on 82-6 hTert and A549 cells exposed to 10 Gy and analyzed 1 h later. p38α activity is measured using the phosphorylation of its target, MK2, on threonine 334 (MK2-pT334) as a proxy. Total MK2 levels are also shown. The protein level of GAPDH, Ku70 and Ku80 serve as loading controls. ( B ) Western blot analysis showing the effect of <t>PF3644022,</t> an inhibitor of MK2 <t>(MK2i),</t> on cells exposed to 10 Gy and analyzed 1 h later. MK2 activity is measured using the phosphorylation of its target, HSP27 at Serine 82 (HSP27-pS82) as a proxy. Total HSP27 levels are also shown. GAPDH serves as a loading control. ( C ) As in A after incubation with 1 µM p38αi or 1 µM MK2i. The ranges of MI values used for normalization are as follows: MI untr = (1.63–2.15%), MI p38αi = (1.81–2.6%) and MI MK2i = (1.37–1.83%). ( D ) Same as in ( C ), but for A549 cells. The ranges of MI values used for normalization are: MI untr = (1.68–2.49%), MI MK2i = (1.84–2.39%) and MI p38αi = (1.66–2.08%). The results in ( C , D ) represent the mean and standard deviation (±SD) from three independent experiments.
    Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    pf3644022 - by Bioz Stars, 2023-12
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    1) Product Images from "The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G 2 -Checkpoint Activation in Cells Exposed to Ionizing Radiation"

    Article Title: The p38/MK2 Pathway Functions as Chk1-Backup Downstream of ATM/ATR in G 2 -Checkpoint Activation in Cells Exposed to Ionizing Radiation

    Journal: Cells

    doi: 10.3390/cells12101387

    Inhibition of p38α or MK2 leaves the G 2 -checkpoint intact in irradiated 82-6 hTert and A549 cells . ( A ) Western blot analysis showing the effect of increasing concentrations of LY2228820, an inhibitor of p38α (p38αi), on 82-6 hTert and A549 cells exposed to 10 Gy and analyzed 1 h later. p38α activity is measured using the phosphorylation of its target, MK2, on threonine 334 (MK2-pT334) as a proxy. Total MK2 levels are also shown. The protein level of GAPDH, Ku70 and Ku80 serve as loading controls. ( B ) Western blot analysis showing the effect of PF3644022, an inhibitor of MK2 (MK2i), on cells exposed to 10 Gy and analyzed 1 h later. MK2 activity is measured using the phosphorylation of its target, HSP27 at Serine 82 (HSP27-pS82) as a proxy. Total HSP27 levels are also shown. GAPDH serves as a loading control. ( C ) As in A after incubation with 1 µM p38αi or 1 µM MK2i. The ranges of MI values used for normalization are as follows: MI untr = (1.63–2.15%), MI p38αi = (1.81–2.6%) and MI MK2i = (1.37–1.83%). ( D ) Same as in ( C ), but for A549 cells. The ranges of MI values used for normalization are: MI untr = (1.68–2.49%), MI MK2i = (1.84–2.39%) and MI p38αi = (1.66–2.08%). The results in ( C , D ) represent the mean and standard deviation (±SD) from three independent experiments.
    Figure Legend Snippet: Inhibition of p38α or MK2 leaves the G 2 -checkpoint intact in irradiated 82-6 hTert and A549 cells . ( A ) Western blot analysis showing the effect of increasing concentrations of LY2228820, an inhibitor of p38α (p38αi), on 82-6 hTert and A549 cells exposed to 10 Gy and analyzed 1 h later. p38α activity is measured using the phosphorylation of its target, MK2, on threonine 334 (MK2-pT334) as a proxy. Total MK2 levels are also shown. The protein level of GAPDH, Ku70 and Ku80 serve as loading controls. ( B ) Western blot analysis showing the effect of PF3644022, an inhibitor of MK2 (MK2i), on cells exposed to 10 Gy and analyzed 1 h later. MK2 activity is measured using the phosphorylation of its target, HSP27 at Serine 82 (HSP27-pS82) as a proxy. Total HSP27 levels are also shown. GAPDH serves as a loading control. ( C ) As in A after incubation with 1 µM p38αi or 1 µM MK2i. The ranges of MI values used for normalization are as follows: MI untr = (1.63–2.15%), MI p38αi = (1.81–2.6%) and MI MK2i = (1.37–1.83%). ( D ) Same as in ( C ), but for A549 cells. The ranges of MI values used for normalization are: MI untr = (1.68–2.49%), MI MK2i = (1.84–2.39%) and MI p38αi = (1.66–2.08%). The results in ( C , D ) represent the mean and standard deviation (±SD) from three independent experiments.

    Techniques Used: Inhibition, Irradiation, Western Blot, Activity Assay, Incubation, Standard Deviation

    Combined inhibition of Chk1 and p38α or MK2 abrogates the G 2 -checkpoint of 82-6 hTert and A549 cells exposed to 2 Gy or IR. ( A ) As in A for 82-6 hTert cells either left untreated or incubated with Chk1i, alone or in combination with p38αi. The ranges of MI used for normalization are: M untr = (1.64–2.28%), MI Chk1i = (1.85–2.56%), and MI Chk1i + p38αi = (2.32–3.46%). ( B ) Same as ( A ), but for A549 cells. The ranges of MI used for normalization are: MI untr = (1.69–3.23%), MI Chki = (2.01–2.49%), and MI Chk1i + p38αi = (2.02–2.88%). ( C ) As in ( A ), but for 82-6 hTert cells treated with combined Chk1i and MK2i. The ranges of MI used for normalization are: M untr = (1.61–2.15%), and MI Chk1i + MK2i = (2.1–2.9%). The red dashed line represents the Chk1i results in ( A ). ( D ) Same as ( B ), but for A549 cells treated with combined Chk1i and MK2i. The ranges of MI used for normalization are: MI untr = (1.72–2.91%) and MI Chk1i + MK2i = (1.87–3.07%). The red dashed line represents Chk1i results, shown in ( B ). All inhibitors were administrated to the cells 1 h prior to irradiation, at the following concentrations: Chk1i, 300 nM, p38αi,1 µM, and MK2i, 1 µM. Data represent mean and standard deviation (±SD) from three independent experiments.
    Figure Legend Snippet: Combined inhibition of Chk1 and p38α or MK2 abrogates the G 2 -checkpoint of 82-6 hTert and A549 cells exposed to 2 Gy or IR. ( A ) As in A for 82-6 hTert cells either left untreated or incubated with Chk1i, alone or in combination with p38αi. The ranges of MI used for normalization are: M untr = (1.64–2.28%), MI Chk1i = (1.85–2.56%), and MI Chk1i + p38αi = (2.32–3.46%). ( B ) Same as ( A ), but for A549 cells. The ranges of MI used for normalization are: MI untr = (1.69–3.23%), MI Chki = (2.01–2.49%), and MI Chk1i + p38αi = (2.02–2.88%). ( C ) As in ( A ), but for 82-6 hTert cells treated with combined Chk1i and MK2i. The ranges of MI used for normalization are: M untr = (1.61–2.15%), and MI Chk1i + MK2i = (2.1–2.9%). The red dashed line represents the Chk1i results in ( A ). ( D ) Same as ( B ), but for A549 cells treated with combined Chk1i and MK2i. The ranges of MI used for normalization are: MI untr = (1.72–2.91%) and MI Chk1i + MK2i = (1.87–3.07%). The red dashed line represents Chk1i results, shown in ( B ). All inhibitors were administrated to the cells 1 h prior to irradiation, at the following concentrations: Chk1i, 300 nM, p38αi,1 µM, and MK2i, 1 µM. Data represent mean and standard deviation (±SD) from three independent experiments.

    Techniques Used: Inhibition, Incubation, Irradiation, Standard Deviation

    Chk2i fails to enhance the G 2 -checkpoint effects of Chk1i, p38αi or MK2i. ( A ) 82-6 hTert cells treated with Chk2i alone or combined with Chk2i and/or p38αi. The MI ranges used for normalization are: MI untr = (1.65–2.17%) and MIChk2i + MK2i = (1.63–1.98%). The green dashed line represents Chk2i data plotted in B. ( B ) 82-6 hTert cells treated with a combination of Chk2i and MK2i. The MI ranges used for normalization are: MI untr = (1.63–2.38%) and MI Chk2i + p38αi = (1.97–2.28%). The green dashed line represents Chk2i data shown in B. ( C ) 82-6 hTert cells treated with a combination of Chk1i, Chk2i and p38αi. The MI ranges used for normalization are: MI untr = (1.58–2.18%) and MI Chk1i + Chk2i + p38αi = (1.98–2.94%). The green dashed line represents the results shown in A. ( D ) 82-hTert cells treated with combinations of Chk1i, Chk2i and MK2i. The MI ranges used for normalization are: MI untr = (1.61–2.15%) and MI Chk1i + Chk2i + MK2i = (1.83–2.91%). The green dashed line represents the results of combined Chk1i and p38αi treatment shown in C. Data represent mean and standard deviation (±SD) from three independent experiments.
    Figure Legend Snippet: Chk2i fails to enhance the G 2 -checkpoint effects of Chk1i, p38αi or MK2i. ( A ) 82-6 hTert cells treated with Chk2i alone or combined with Chk2i and/or p38αi. The MI ranges used for normalization are: MI untr = (1.65–2.17%) and MIChk2i + MK2i = (1.63–1.98%). The green dashed line represents Chk2i data plotted in B. ( B ) 82-6 hTert cells treated with a combination of Chk2i and MK2i. The MI ranges used for normalization are: MI untr = (1.63–2.38%) and MI Chk2i + p38αi = (1.97–2.28%). The green dashed line represents Chk2i data shown in B. ( C ) 82-6 hTert cells treated with a combination of Chk1i, Chk2i and p38αi. The MI ranges used for normalization are: MI untr = (1.58–2.18%) and MI Chk1i + Chk2i + p38αi = (1.98–2.94%). The green dashed line represents the results shown in A. ( D ) 82-hTert cells treated with combinations of Chk1i, Chk2i and MK2i. The MI ranges used for normalization are: MI untr = (1.61–2.15%) and MI Chk1i + Chk2i + MK2i = (1.83–2.91%). The green dashed line represents the results of combined Chk1i and p38αi treatment shown in C. Data represent mean and standard deviation (±SD) from three independent experiments.

    Techniques Used: Standard Deviation

    Three parametric flow cytometry analysis of G 2 -checkpoint confirms a p38α and MK2 dependent checkpoint activation when analyzing exclusively G 2 -phase irradiated cells after Chk1 inhibition. ( A ) Three parametric flow cytometry analysis showing the normalized MI as a function of time in EdU − , G 2 -cells, in non-irradiated or 2 Gy irradiated 82-6 hTert cells. ( B ) As in ( A ) for samples treated with Chk1i. The dashed lines depict the results shown in ( A ) for comparison. ( C ) As in ( B ) for samples treated with both Chk1i and p38αi. The dashed lines are transferred from ( A , B ) and are shown for comparison. ( D ) As in ( C ), but for cells irradiated in the presence of both Chk1i and MK2i. See text for details on methodology and interpretation. Data represent mean and standard deviation (±SD) from three independent experiments.
    Figure Legend Snippet: Three parametric flow cytometry analysis of G 2 -checkpoint confirms a p38α and MK2 dependent checkpoint activation when analyzing exclusively G 2 -phase irradiated cells after Chk1 inhibition. ( A ) Three parametric flow cytometry analysis showing the normalized MI as a function of time in EdU − , G 2 -cells, in non-irradiated or 2 Gy irradiated 82-6 hTert cells. ( B ) As in ( A ) for samples treated with Chk1i. The dashed lines depict the results shown in ( A ) for comparison. ( C ) As in ( B ) for samples treated with both Chk1i and p38αi. The dashed lines are transferred from ( A , B ) and are shown for comparison. ( D ) As in ( C ), but for cells irradiated in the presence of both Chk1i and MK2i. See text for details on methodology and interpretation. Data represent mean and standard deviation (±SD) from three independent experiments.

    Techniques Used: Flow Cytometry, Activation Assay, Irradiation, Inhibition, Standard Deviation

    Three parametric flow cytometry analysis of G 2 -checkpoint reveals a p38α/MK2 dependent activation also in S-phase irradiated cells following Chk1 inhibition. ( A ) Percentage of EdU + cells, representing the fraction of S-phase cells at the time of irradiation with 2 Gy, entering the G 2 -phase as a function of time thereafter. Results with unirradiated cells are also shown. The dashed line is arbitrarily drawn at 20% to help estimate the radiation-induced delay. ( B ) As in ( A ) for cells treated with Chk1i. The dashed lines depict the results in ( A ) and are shown for comparison. ( C ) As in ( B ) for cells treated with both Chk1i and p38αi. ( D ) As in ( C ) for cells treated with Chk1i and MK2i. See text for details on methodology and interpretation. Data represent mean and standard deviation (±SD) from three independent experiments.
    Figure Legend Snippet: Three parametric flow cytometry analysis of G 2 -checkpoint reveals a p38α/MK2 dependent activation also in S-phase irradiated cells following Chk1 inhibition. ( A ) Percentage of EdU + cells, representing the fraction of S-phase cells at the time of irradiation with 2 Gy, entering the G 2 -phase as a function of time thereafter. Results with unirradiated cells are also shown. The dashed line is arbitrarily drawn at 20% to help estimate the radiation-induced delay. ( B ) As in ( A ) for cells treated with Chk1i. The dashed lines depict the results in ( A ) and are shown for comparison. ( C ) As in ( B ) for cells treated with both Chk1i and p38αi. ( D ) As in ( C ) for cells treated with Chk1i and MK2i. See text for details on methodology and interpretation. Data represent mean and standard deviation (±SD) from three independent experiments.

    Techniques Used: Flow Cytometry, Activation Assay, Irradiation, Inhibition, Standard Deviation

    Roles of ATM and ATR in the activation of p38α/MK2 pathway and the G 2 -checkpoint. ( A ) Western blot analysis of pMK2-T334 and pHSP27-S82 after treatment of 82-6 hTert cells with the indicated PIKK inhibitors. GAPDH, HSP27, Ku80 and Chk1 served as loading controls. ( B ) G 2 -checkpoint analysis in 82-6 hTert cells treated with ATRi, or a combination of ATRi and p38αi. The range of MI are: MI untr = (1.61–2.14%), MI ATRi = (2–2.92%), and MI ATRi + p38αi = (2.02–2.88%). ( C ) G 2 -checkpoint analysis in 82-6 hTert cells treated with a combination of ATRi and MK2i. The ranges of MI are MI ATRi + MK2i = (1.6–2.87%). ( D ) G 2 -checkpoint analysis in 82-6 hTert cells treated with Ku55933 (ATMi) or a combination of ATMi and p38αi. The ranges of MI are: MI untr = (1.61–2.14%), MI ATMi = (1.68–1.84%), and MI ATMi + p38αi = (1.76–2.93%). ( E ) G 2 -checkpoint analysis in 82-6 hTert cells treated with a combination of ATMi and MK2i. The range of MI is MI ATMi + MK2i = (1.78–2.67%). Dashed lines in ( C , E ) illustrate data presented in ( B , D ), respectively, and are shown for comparison. All data represent means and standard deviation (±SD) from two or three independent experiments.
    Figure Legend Snippet: Roles of ATM and ATR in the activation of p38α/MK2 pathway and the G 2 -checkpoint. ( A ) Western blot analysis of pMK2-T334 and pHSP27-S82 after treatment of 82-6 hTert cells with the indicated PIKK inhibitors. GAPDH, HSP27, Ku80 and Chk1 served as loading controls. ( B ) G 2 -checkpoint analysis in 82-6 hTert cells treated with ATRi, or a combination of ATRi and p38αi. The range of MI are: MI untr = (1.61–2.14%), MI ATRi = (2–2.92%), and MI ATRi + p38αi = (2.02–2.88%). ( C ) G 2 -checkpoint analysis in 82-6 hTert cells treated with a combination of ATRi and MK2i. The ranges of MI are MI ATRi + MK2i = (1.6–2.87%). ( D ) G 2 -checkpoint analysis in 82-6 hTert cells treated with Ku55933 (ATMi) or a combination of ATMi and p38αi. The ranges of MI are: MI untr = (1.61–2.14%), MI ATMi = (1.68–1.84%), and MI ATMi + p38αi = (1.76–2.93%). ( E ) G 2 -checkpoint analysis in 82-6 hTert cells treated with a combination of ATMi and MK2i. The range of MI is MI ATMi + MK2i = (1.78–2.67%). Dashed lines in ( C , E ) illustrate data presented in ( B , D ), respectively, and are shown for comparison. All data represent means and standard deviation (±SD) from two or three independent experiments.

    Techniques Used: Activation Assay, Western Blot, Standard Deviation

    mk2 inhibitor pf3644022  (Tocris)

     
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    Tocris mk2 inhibitor pf3644022
    Mk2 Inhibitor Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pf3644022  (Tocris)

     
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    pf3644022  (Tocris)

     
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    Tocris pf3644022
    Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pf3644022  (Tocris)

     
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    Tocris pf3644022
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    mk2 inhibitor pf3644022  (Tocris)

     
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    Tocris mk2 inhibitor pf3644022
    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 <t>MK2</t> (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 <t>MAPKAPK2</t> 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.
    Mk2 Inhibitor Pf3644022, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13- cis RA-mediated differentiation in neuroblastoma"

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

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2563-4

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    mk2 inhibitor pf3644022  (Tocris)

     
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