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gsk2830371  (MedChemExpress)
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MedChemExpress gsk2830371
A Parental RPE, <t>RPE-PPM1D-T1</t> and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) in the presence or absence of PPM1Di and further cultured for 10 d. Surviving fraction was calculated by normalising the colony number to the non-treated control for each genotype. Error bars indicate SD. Statistical significance was determined by Student’s t test (*p ≤ 0.05, n = 3). B Parental RPE, RPE-PPM1D-T1 and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) in presence or absence of PPM1Di and fixed after 48 h. Cells were then stained with DAPI and percentage of cells containing micronuclei was determined microscopically. More than 200 cells per condition were quantified in each experiment (n = 3), error bars indicate SD. Statistical significance was determined by Student’s t test (*p ≤ 0.05) C Parental RPE and RPE-PPM1D-T2 cells were stably transfected RFP-cGAS and were fixed 48 h after mock treatment or irradiation (3 Gy). Note accumulation of RFP-cGAS in MNs in cells exposed to IR. D Parental RPE and RPE-PPM1D-T2 stably transfected with RFP-cGAS were mock treated or irradiated (3 Gy) in presence or absence of PPM1Di. Whole cell lysates were collected after 48 h and analysed by immunoblotting. Asterisk indicates a non-specific reactivity. Signal of IRF3-pSer386 was quantified in ImageJ from 3 independent repeats and was normalised to the loading control (TFIIH) and to the non-treated condition. Statistical significance was determined by Student’s t test (*p ≤ 0.05, n = 3, error bars indicate SD). E RNA was collected from cells grown as in ( D ) and expression of indicated genes was analysed by qPCR. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001).
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A Parental RPE, RPE-PPM1D-T1 and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) in the presence or absence of PPM1Di and further cultured for 10 d. Surviving fraction was calculated by normalising the colony number to the non-treated control for each genotype. Error bars indicate SD. Statistical significance was determined by Student’s t test (*p ≤ 0.05, n = 3). B Parental RPE, RPE-PPM1D-T1 and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) in presence or absence of PPM1Di and fixed after 48 h. Cells were then stained with DAPI and percentage of cells containing micronuclei was determined microscopically. More than 200 cells per condition were quantified in each experiment (n = 3), error bars indicate SD. Statistical significance was determined by Student’s t test (*p ≤ 0.05) C Parental RPE and RPE-PPM1D-T2 cells were stably transfected RFP-cGAS and were fixed 48 h after mock treatment or irradiation (3 Gy). Note accumulation of RFP-cGAS in MNs in cells exposed to IR. D Parental RPE and RPE-PPM1D-T2 stably transfected with RFP-cGAS were mock treated or irradiated (3 Gy) in presence or absence of PPM1Di. Whole cell lysates were collected after 48 h and analysed by immunoblotting. Asterisk indicates a non-specific reactivity. Signal of IRF3-pSer386 was quantified in ImageJ from 3 independent repeats and was normalised to the loading control (TFIIH) and to the non-treated condition. Statistical significance was determined by Student’s t test (*p ≤ 0.05, n = 3, error bars indicate SD). E RNA was collected from cells grown as in ( D ) and expression of indicated genes was analysed by qPCR. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001).

Journal: Oncogene

Article Title: PPM1D activity promotes cellular transformation by preventing senescence and cell death

doi: 10.1038/s41388-024-03149-3

Figure Lengend Snippet: A Parental RPE, RPE-PPM1D-T1 and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) in the presence or absence of PPM1Di and further cultured for 10 d. Surviving fraction was calculated by normalising the colony number to the non-treated control for each genotype. Error bars indicate SD. Statistical significance was determined by Student’s t test (*p ≤ 0.05, n = 3). B Parental RPE, RPE-PPM1D-T1 and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) in presence or absence of PPM1Di and fixed after 48 h. Cells were then stained with DAPI and percentage of cells containing micronuclei was determined microscopically. More than 200 cells per condition were quantified in each experiment (n = 3), error bars indicate SD. Statistical significance was determined by Student’s t test (*p ≤ 0.05) C Parental RPE and RPE-PPM1D-T2 cells were stably transfected RFP-cGAS and were fixed 48 h after mock treatment or irradiation (3 Gy). Note accumulation of RFP-cGAS in MNs in cells exposed to IR. D Parental RPE and RPE-PPM1D-T2 stably transfected with RFP-cGAS were mock treated or irradiated (3 Gy) in presence or absence of PPM1Di. Whole cell lysates were collected after 48 h and analysed by immunoblotting. Asterisk indicates a non-specific reactivity. Signal of IRF3-pSer386 was quantified in ImageJ from 3 independent repeats and was normalised to the loading control (TFIIH) and to the non-treated condition. Statistical significance was determined by Student’s t test (*p ≤ 0.05, n = 3, error bars indicate SD). E RNA was collected from cells grown as in ( D ) and expression of indicated genes was analysed by qPCR. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001).

Article Snippet: GSK2830371 (referred to as PPM1D inhibitor; final concentration 2 μM, [ , ]) and MDM2 antagonist nutlin-3 (final concentration 9 μM, [ ]) and caspase inhibitor Z-VAD-FMK (final concentration 10 μM, [ ]) were from MedchemExpress (Monmouth Junction, NJ) and were dissolved in DMSO.

Techniques: Irradiation, Cell Culture, Control, Staining, Stable Transfection, Transfection, Western Blot, Expressing

A Parental RPE and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) and were grown in semi-solid media for 12 weeks. Six independent clones of RPE-PPM1D-T2-SA cells were collected. B Colony size of parental RPE cells, parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2 clones was acquired after propagating in the semisolid media for 2 weeks. Each dot represents a single colony, red line indicates mean colony size, bars show SD, n = 2. C Cell division was monitored by labelling of cells with CFSE. A zero time point was collected to determine the initial labelling and the rest of the samples were collected after 48 h. Cell were then collected and fixed and analysed by FACS. Plotted is the mean intensity of CFSE signal, error bars indicate SD, n = 3. Statistical significance was calculated using Student’s t test (*p ≤ 0.05, **p ≤ 0.01). D Cell cycle distribution was determined in parental RPE, parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2 clones using flow cytometry. Plotted are fractions of cells in G1, S, G2 and M phases of the cell cycle. Error bars indicate SDs, n = 3. E Nude mice were subcutaneously injected with parental RPE cells, parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2 clones and tumour growth was evaluated after 3 weeks.

Journal: Oncogene

Article Title: PPM1D activity promotes cellular transformation by preventing senescence and cell death

doi: 10.1038/s41388-024-03149-3

Figure Lengend Snippet: A Parental RPE and RPE-PPM1D-T2 cells were mock treated or irradiated (3 Gy) and were grown in semi-solid media for 12 weeks. Six independent clones of RPE-PPM1D-T2-SA cells were collected. B Colony size of parental RPE cells, parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2 clones was acquired after propagating in the semisolid media for 2 weeks. Each dot represents a single colony, red line indicates mean colony size, bars show SD, n = 2. C Cell division was monitored by labelling of cells with CFSE. A zero time point was collected to determine the initial labelling and the rest of the samples were collected after 48 h. Cell were then collected and fixed and analysed by FACS. Plotted is the mean intensity of CFSE signal, error bars indicate SD, n = 3. Statistical significance was calculated using Student’s t test (*p ≤ 0.05, **p ≤ 0.01). D Cell cycle distribution was determined in parental RPE, parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2 clones using flow cytometry. Plotted are fractions of cells in G1, S, G2 and M phases of the cell cycle. Error bars indicate SDs, n = 3. E Nude mice were subcutaneously injected with parental RPE cells, parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2 clones and tumour growth was evaluated after 3 weeks.

Article Snippet: GSK2830371 (referred to as PPM1D inhibitor; final concentration 2 μM, [ , ]) and MDM2 antagonist nutlin-3 (final concentration 9 μM, [ ]) and caspase inhibitor Z-VAD-FMK (final concentration 10 μM, [ ]) were from MedchemExpress (Monmouth Junction, NJ) and were dissolved in DMSO.

Techniques: Irradiation, Clone Assay, Transformation Assay, Flow Cytometry, Injection

A Parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2-SA (clones 1 and 6) were arrested in mitosis, fixed and probed by M-FISH. Karyotyping of the remaining clones is shown in Suppl. Figure . B Selected genes differentially expressed in parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA cells. The heat map shows the differential expression normalised to nontransformed parental RPE-PPM1D-T2 cells. C Asynchronously growing parental RPE, RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA clones 1–6 were lysed and expression of selected proteins was determined by immunoblotting. TFIIH and 14-3-3 were used as loading controls. Arrowhead indicates the position of PIK3IP protein. D Parental RPE, RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA clones 1–6 were exposed or not to a high dose of IR (5 Gy), collected after 6 h and whole cell lysates were analysed by immunoblotting.

Journal: Oncogene

Article Title: PPM1D activity promotes cellular transformation by preventing senescence and cell death

doi: 10.1038/s41388-024-03149-3

Figure Lengend Snippet: A Parental RPE-PPM1D-T2 cells and transformed RPE-PPM1D-T2-SA (clones 1 and 6) were arrested in mitosis, fixed and probed by M-FISH. Karyotyping of the remaining clones is shown in Suppl. Figure . B Selected genes differentially expressed in parental RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA cells. The heat map shows the differential expression normalised to nontransformed parental RPE-PPM1D-T2 cells. C Asynchronously growing parental RPE, RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA clones 1–6 were lysed and expression of selected proteins was determined by immunoblotting. TFIIH and 14-3-3 were used as loading controls. Arrowhead indicates the position of PIK3IP protein. D Parental RPE, RPE-PPM1D-T2 and transformed RPE-PPM1D-T2-SA clones 1–6 were exposed or not to a high dose of IR (5 Gy), collected after 6 h and whole cell lysates were analysed by immunoblotting.

Article Snippet: GSK2830371 (referred to as PPM1D inhibitor; final concentration 2 μM, [ , ]) and MDM2 antagonist nutlin-3 (final concentration 9 μM, [ ]) and caspase inhibitor Z-VAD-FMK (final concentration 10 μM, [ ]) were from MedchemExpress (Monmouth Junction, NJ) and were dissolved in DMSO.

Techniques: Transformation Assay, Clone Assay, Expressing, Western Blot

A BJ-hTert-HRASV12 ER-TAM and two clones expanded after targeting the exon 6 of PPM1D by CRISPR/Cas9 were analysed by immunoblotting. Staining for actin was used as loading control. B BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 cells were induced with 4OHT for indicated times and whole cell lysates were analysed by immunoblotting. Staining for 14-3-3 protein was used as loading control. C BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 were induced with 4OHT for 2 d and were labelled with CldU and IdU 20 min. Plotted is the sum of the length (μm) of CldU and IdU labelled tracks, each dot represents one replication fork, n = 3. Black line indicates mean. Statistical significance was calculated by Student’s t test (****p ≤ 0.0001). D BJ-hTert-HRASV12 ER-TAM and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 cells were induced with 4OHT for 5 d and formation of 53BP1 nuclear foci was evaluated by ScanR microscopy. Plotted is a fraction of cells with ≥6 53BP1 nuclear foci. More than 300 cells were quantified per experiment, bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). E Cells from ( D ) were probed for γH2AX and analyzed by ScanR microscopy. Plotted is mean nuclear signal of γH2AX in G1 cells. Statistical significance was calculated by Student’s t test (**p ≤ 0.01, ***p ≤ 0.001). F BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 induced with 4OHT for 5 d. Fraction of cells containing MN was determined microscopically. Bars indicate SD, more than 150 cells per condition were quantified in each experiment, n = 3. Statistical significance was calculated by Student’s t test (ns p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Journal: Oncogene

Article Title: PPM1D activity promotes cellular transformation by preventing senescence and cell death

doi: 10.1038/s41388-024-03149-3

Figure Lengend Snippet: A BJ-hTert-HRASV12 ER-TAM and two clones expanded after targeting the exon 6 of PPM1D by CRISPR/Cas9 were analysed by immunoblotting. Staining for actin was used as loading control. B BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 cells were induced with 4OHT for indicated times and whole cell lysates were analysed by immunoblotting. Staining for 14-3-3 protein was used as loading control. C BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 were induced with 4OHT for 2 d and were labelled with CldU and IdU 20 min. Plotted is the sum of the length (μm) of CldU and IdU labelled tracks, each dot represents one replication fork, n = 3. Black line indicates mean. Statistical significance was calculated by Student’s t test (****p ≤ 0.0001). D BJ-hTert-HRASV12 ER-TAM and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 cells were induced with 4OHT for 5 d and formation of 53BP1 nuclear foci was evaluated by ScanR microscopy. Plotted is a fraction of cells with ≥6 53BP1 nuclear foci. More than 300 cells were quantified per experiment, bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). E Cells from ( D ) were probed for γH2AX and analyzed by ScanR microscopy. Plotted is mean nuclear signal of γH2AX in G1 cells. Statistical significance was calculated by Student’s t test (**p ≤ 0.01, ***p ≤ 0.001). F BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 induced with 4OHT for 5 d. Fraction of cells containing MN was determined microscopically. Bars indicate SD, more than 150 cells per condition were quantified in each experiment, n = 3. Statistical significance was calculated by Student’s t test (ns p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Article Snippet: GSK2830371 (referred to as PPM1D inhibitor; final concentration 2 μM, [ , ]) and MDM2 antagonist nutlin-3 (final concentration 9 μM, [ ]) and caspase inhibitor Z-VAD-FMK (final concentration 10 μM, [ ]) were from MedchemExpress (Monmouth Junction, NJ) and were dissolved in DMSO.

Techniques: Clone Assay, CRISPR, Western Blot, Staining, Control, Microscopy

A BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 cells were induced or not with 4OHT for 20 d and stained for β-galactosidase activity. Plotted is the fraction of β-gal positive cells, bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (***p ≤ 0.001, ****p ≤ 0.0001). B Cells grown as in A were fixed and stained for p16. Plotted is the mean nuclear p16 intensity, bars indicate SD. More than 300 cells of each condition were quantified per experiment, n = 3. Statistical significance was calculated by Student’s t test (**p ≤ 0.01, ***p ≤ 0.001). C Cells as in A were induced with 4OHT for 5 to 20 days were incubated with EdU 24 h prior fixation followed by CLICK-IT reaction. Fraction of EdU positive cells was analysed using FACS. Bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). D Whole cell lysates from cells from ( C ) were analyzed by immunoblotting using indicated antibodies. E BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and -T2 cells were treated or not with 4OHT for 5 days. Whole cell lysates were probed with indicated antibodies by immunoblotting. F Cells treated as in ( E ) were analysed by flow cytometry. Where indicated, cells were incubated in the presence of Z-VAD-FMK. Plotted is the fraction of cleaved caspase 3 positive cells. Bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test. G Parental BJ-hTert-HRASV12 ER-TAM , parental BJ-hTert-HRASV12 ER-TAM -PPM1D-T2, and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2-60 cells that survived 2-month continuous induction with 4OHT were cultured in semisolid media for 10 weeks.

Journal: Oncogene

Article Title: PPM1D activity promotes cellular transformation by preventing senescence and cell death

doi: 10.1038/s41388-024-03149-3

Figure Lengend Snippet: A BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2 cells were induced or not with 4OHT for 20 d and stained for β-galactosidase activity. Plotted is the fraction of β-gal positive cells, bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (***p ≤ 0.001, ****p ≤ 0.0001). B Cells grown as in A were fixed and stained for p16. Plotted is the mean nuclear p16 intensity, bars indicate SD. More than 300 cells of each condition were quantified per experiment, n = 3. Statistical significance was calculated by Student’s t test (**p ≤ 0.01, ***p ≤ 0.001). C Cells as in A were induced with 4OHT for 5 to 20 days were incubated with EdU 24 h prior fixation followed by CLICK-IT reaction. Fraction of EdU positive cells was analysed using FACS. Bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001). D Whole cell lysates from cells from ( C ) were analyzed by immunoblotting using indicated antibodies. E BJ-hTert-HRASV12 ER-TAM , BJ-hTert-HRASV12 ER-TAM -PPM1D-T1 and -T2 cells were treated or not with 4OHT for 5 days. Whole cell lysates were probed with indicated antibodies by immunoblotting. F Cells treated as in ( E ) were analysed by flow cytometry. Where indicated, cells were incubated in the presence of Z-VAD-FMK. Plotted is the fraction of cleaved caspase 3 positive cells. Bars indicate SD, n = 3. Statistical significance was calculated by Student’s t test. G Parental BJ-hTert-HRASV12 ER-TAM , parental BJ-hTert-HRASV12 ER-TAM -PPM1D-T2, and BJ-hTert-HRASV12 ER-TAM -PPM1D-T2-60 cells that survived 2-month continuous induction with 4OHT were cultured in semisolid media for 10 weeks.

Article Snippet: GSK2830371 (referred to as PPM1D inhibitor; final concentration 2 μM, [ , ]) and MDM2 antagonist nutlin-3 (final concentration 9 μM, [ ]) and caspase inhibitor Z-VAD-FMK (final concentration 10 μM, [ ]) were from MedchemExpress (Monmouth Junction, NJ) and were dissolved in DMSO.

Techniques: Staining, Activity Assay, Incubation, Western Blot, Flow Cytometry, Cell Culture