In Swiss 3T3 fibroblasts, PD98059 pretreatment inhibited PDGF-stimulated MAPK tyrosine phosphorylation with an apparent IC50 of approximately 10 μM and reduced MAP2-phosphorylating activity in cell lysates. [1]
In HL-60 cells, 48-hour PD98059 exposure produced 50% inhibition of viable-cell proliferation at 300 μM, the highest concentration tested. This endpoint does not distinguish reduced division from differentiation or cell death. [2]
Inhibition of MEK-dependent MAPK activation in recombinant assays
Biochemical assay · GST-MEK1, partially active GST-MEK-2E (S218E/S222E), GST-MAPK and kinase-negative GST-MAPK-KA [1]
PD 098059 inhibited basal and partially active MEK-mediated substrate phosphorylation. Order-of-addition experiments distinguished prevention of MAPK activation from inhibition of activated MAPK; washout restored MEK activity.
Literature-Reported Treatment Conditions
- Treatment concentration
- Fig. 2A: 0.1, 1, 10, 100 μM; Fig. 2B: 50 μM; washout experiment: 30 μM
- Exposure duration
- Kinase assay: 15 min at 30°C; Fig. 2C: 30-min compound incubation before washes
- Method
- Radiolabeled phosphorylation of MBP or kinase-negative MAPK; order-of-addition and washout controls
- Reported limits
- Vehicle and replicate n for IC50 not specified in the read assay text; Fig. 2B samples were run in duplicate.
Methods and source locations
Compound reported in the paper: PD98059; Compound label in the paper: PD 098059
Evidence concerns the named research compound; no supplier interchangeability, lot purity, salt conversion or clinical efficacy is established.
pp. 7686–7687, In Vitro Kinase Assay; Fig. 1 structure; Fig. 2A–C and Results
Basal GST-MEK1 and partially active GST-MEK-2E substrate phosphorylation: Phosphate incorporation into MAPK-KA or phosphotyrosine immunoblotting — p. 7687, Results paragraph below Fig. 2
MEK-dependent MAPK activation versus activated MAPK activity: GST-MEK1/GST-MAPK/MBP cascade assay — p. 7687, Fig. 2A and Results
Recovery of MEK activity after removing inhibitor: GST-MEK1 retained on glutathione beads; MAPK-KA phosphorylation assay — pp. 7687–7688, Fig. 2C caption and adjoining Results
Acute PDGF-stimulated MAPK signaling in Swiss 3T3 cells
Cell-based study · Serum-deprived Swiss 3T3 mouse fibroblasts [1]
Pretreatment inhibited PDGF-stimulated MAPK tyrosine phosphorylation and MAP2-phosphorylating activity in cell lysates.
Literature-Reported Treatment Conditions
- Treatment concentration
- Fig. 3A: 1, 3, 6, 10, 25, 50, 100 μM; Fig. 3B: 1, 10, 100 μM
- Exposure duration
- 30-min pretreatment followed by 5-min PDGF stimulation
- Method
- Compound added to serum-deprived cells; subsequent MAPK immunoprecipitation/immunoblot and lysate MAP2 phosphorylation
- Vehicle
- DMSO control shown in Fig. 3B; concentration not specified there
- Co-treatment
- PDGF 100 ng/ml
- Reported limits
- Replicate n and variability for Fig. 3A/B not specified in read text.
Methods and source locations
Compound reported in the paper: PD98059; Compound label in the paper: PD 098059
Evidence concerns the named research compound; no supplier interchangeability, lot purity, salt conversion or clinical efficacy is established.
pp. 7686–7688, Immunoprecipitation/Immunoblot Analysis; MAP2 phosphorylation assay; Fig. 3A/B and Results
PDGF-stimulated MAPK tyrosine phosphorylation: Anti-phosphotyrosine immunoblot of immunoprecipitated MAPK — p. 7688, Fig. 3A and Results
PDGF-induced MAPK activity in whole-cell lysate: Radiolabeled MAP2 phosphorylation; unstimulated, PDGF and DMSO controls shown — p. 7688, Fig. 3B and Results
HL-60 proliferation dose response
Cell-based study · HL-60 human myeloid leukemia cells [2]
PD98059 slowed accumulation of viable HL-60 cells less potently than the other tested MEK inhibitors. The endpoint includes division, differentiation and death rather than measuring any one process alone.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 nM–300 μM
- Exposure duration
- 48 h
- Method
- Viable cell counting by trypan blue exclusion
- Vehicle
- DMSO; controls received corresponding concentration
- Reported limits
- No fitted proliferation IC50 is adopted.
Methods and source locations
Compound reported in the paper: PD98059; Compound label in the paper: PD98059
Evidence concerns the named research compound; no supplier interchangeability, lot purity, salt conversion or clinical efficacy is established.
Experimental Procedures: Cell Culture and Inhibitors; Results first subsection; Fig. 1A and caption
Viable-cell proliferation relative to vehicle-treated control: Trypan blue exclusion; mean ± S.D., n = 3 for Fig. 1; reported significant reduction at ≥30 μM, p < 0.05 — Fig. 1A, caption and Results: Dose-dependent Inhibition of Proliferation
Acute mitochondrial respiratory effects in intact HL-60 cells
Cell-based study · Intact HL-60 cells assessed by multiwavelength spectroscopy and oxygen-consumption measurements [2]
PD98059 caused dose-dependent heme oxidation and reduced oxygen consumption. These cellular findings limit attribution of high-concentration responses solely to MEK inhibition. PD98059 interference prevented determination of its NAD(H) response.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1–100 μM
- Exposure duration
- Measurements 2 min after treatment
- Method
- Multiwavelength heme spectroscopy with simultaneous oxygen consumption; DMSO concentration controls
- Vehicle
- DMSO; corresponding concentration controls
- Reported limits
- PD98059 NAD(H) effect could not be determined because the compound fluoresced; no direct PD98059–complex I binding assay is established.
Methods and source locations
Compound reported in the paper: PD98059; Compound label in the paper: PD98059
Evidence concerns the named research compound; no supplier interchangeability, lot purity, salt conversion or clinical efficacy is established.
Experimental Procedures: Inhibitors and Cell Spectroscopy/Analysis; Results: Direct Effects on ETC; Fig. 3 and caption
Oxygen consumption and cytochrome oxidation: Fig. 3: mean ± S.D., n = 6; oxygen consumption as percentage of baseline; heme oxidation assessed spectroscopically — Fig. 3 caption, PD98059 panels and Results: Direct Effects on ETC
NAD(H) oxidation response to PD98059: NADH fluorometry; limitation explicitly stated in Results — Results: Direct Effects on ETC, paragraph after Fig. 3
