PLX4032 inhibited growth of the tested BRAF V600E melanoma panel more strongly than BRAF wild-type lines. The authors reported an approximate cellular IC50 of ~5 µM for their small mutant panel and noted higher values than in other reports, illustrating model-dependent sensitivity. [2]
In G361 and SK-MEL-2 cells, vemurafenib reduced MTT viability with increasing concentration. Dock180 and/or Elmo1 knockdown was associated with additional viability loss, including without vemurafenib; these findings do not demonstrate drug–siRNA synergy. [5]
Biochemical RAF inhibition and kinase selectivity
Biochemical assay · BRAF V600E, wild-type BRAF, CRAF and counter-screened kinases [1]
PLX4032 inhibited BRAF V600E but also CRAF and other kinases. The RAF comparison used 100 µM ATP; most counter-screens used 10 µM ATP, limiting direct cross-kinase selectivity comparisons.
Literature-Reported Treatment Conditions
- Method
- Biochemical kinase activity IC50 determinations; assay implementation referenced to earlier work in Methods Summary and SI.
- Reported limits
- Dose series, reaction duration, replicate counts and uncertainty are not specified in the reviewed main/SI assay summary and Table 1. Earlier referenced protocol not acquired; no conditions imported.
Methods and source locations
Compound reported in the paper: PLX4032 (vemurafenib); Compound label in the paper: PLX4032
Literature evidence for the named compound; tested material does not validate an Adooq lot.
Main text kinase-selectivity paragraph; SI Preclinical studies p.4, kinase-selectivity Results p.6 and Supplementary Table 1 p.9
BRAF V600E kinase inhibition: Biochemical activity assay at 100 µM ATP; Table 1 reports point value without n or error. — Main text kinase-selectivity paragraph; SI Preclinical studies p.4, kinase-selectivity Results p.6 and Supplementary Table 1 p.9
CRAF kinase inhibition: Biochemical activity assay at 100 µM ATP. — Main text kinase-selectivity paragraph; SI Preclinical studies p.4, kinase-selectivity Results p.6 and Supplementary Table 1 p.9
Wild-type BRAF kinase inhibition: Biochemical activity assay at 100 µM ATP. — Main text kinase-selectivity paragraph; SI Preclinical studies p.4, kinase-selectivity Results p.6 and Supplementary Table 1 p.9
Selected non-RAF kinase inhibition: SRMS at 100 µM ATP; ACK1, MAP4K5 and FGR at 10 µM ATP. Biochemical potency does not establish cellular off-target engagement. — Main text kinase-selectivity paragraph; SI Preclinical studies p.4, kinase-selectivity Results p.6 and Supplementary Table 1 p.9
Genotype-dependent ERK phosphorylation response
Cell-based study · WM35, WM793 and 1205Lu (BRAF V600E); WM852 and SbCln2 (BRAF wild-type/NRAS mutant); C8161 (BRAF/NRAS wild-type) [2]
After 2 h, PLX4032 reduced pERK in the tested BRAF V600E lines. The wild-type BRAF lines did not show the same inhibitory pattern. β-actin was the loading control.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 nM, 1 µM and 10 µM; untreated comparison
- Exposure duration
- 2 h
- Method
- Immunoblotting of pERK; β-actin loading control.
- Reported limits
- Drug vehicle and biological replicate count are not specified for Figure 1 in the reviewed methods/caption.
Methods and source locations
Compound reported in the paper: PLX4032 (vemurafenib); Compound label in the paper: PLX4032
Literature evidence for the named compound; tested material does not validate an Adooq lot.
Table 1; Figure 1 image/caption; Specificity of MAPK Inhibition; Western Blot Analysis methods
ERK phosphorylation: pERK immunoblot with β-actin loading control, 2 h. — Table 1; Figure 1 image/caption; Specificity of MAPK Inhibition; Western Blot Analysis methods
Cellular growth inhibition varies with model
Cell-based study · BRAF V600E WM35, WM793, 1205Lu versus BRAF wild-type SbCln2, C8161, WM1361A melanoma cells [2]
Growth was inhibited in the tested mutant panel, whereas wild-type BRAF lines were comparatively insensitive. The authors describe an approximate IC50 of ~5 µM for their small mutant panel, above values reported elsewhere; they discuss PTEN alterations as a possible explanation rather than proving causation.
Literature-Reported Treatment Conditions
- Treatment concentration
- Increasing concentrations; Figure 2A axis extends to 5000 nM.
- Exposure duration
- 72 h drug exposure
- Method
- Tetrazolium-based proliferation assay; Methods name MTT, whereas Results name MTS. Absorbance normalized against untreated growth after initial absorbance subtraction.
- Reported limits
- Drug vehicle is not specified in the proliferation methods. Assay identity is inconsistent (MTT versus MTS); no exact cell-line IC50 fitted or read from curves.
Methods and source locations
Compound reported in the paper: PLX4032 (vemurafenib); Compound label in the paper: PLX4032
Literature evidence for the named compound; tested material does not validate an Adooq lot.
Cell Proliferation Assays methods; Figure 2A image/caption; Selectivity Profile Results; Discussion paragraph beginning Our small panel
Growth inhibition in the small BRAF V600E melanoma panel: Author-stated estimate in Discussion, not an exact curve-derived value. Methods specify quadruplicate wells and mean of at least three independent experiments ± SE; no uncertainty is attached to this IC50 estimate. — Cell Proliferation Assays methods; Figure 2A image/caption; Selectivity Profile Results; Discussion paragraph beginning Our small panel
Vemurafenib response with Dock180/Elmo1 knockdown
Cell-based study · Human G361 and SK-MEL-2 melanoma cells with siRNA control, Dock180 siRNA, Elmo1 siRNA or both [5]
Vemurafenib reduced MTT viability with increasing concentration. Dock180 and/or Elmo1 knockdown was associated with lower viability, including at zero vemurafenib. This comparison does not establish pharmacological synergy or that vemurafenib alone causes the siRNA-associated effects.
Literature-Reported Treatment Conditions
- Treatment concentration
- Vemurafenib: 0, 0.5, 1 and 1.5 µM (Figure 6).
- Method
- MTT cell viability assay; Figure 6 data are mean±SD of three independent experiments.
- Co-treatment
- 20 nM Dock180- and/or Elmo1-specific siRNA; Stealth siRNA control. Generic Methods do not unambiguously state total siRNA concentration in the double-knockdown arm.
- Reported limits
- Generic Methods list 24, 48 or 72 h but Figure 6 does not identify a specific duration. Vemurafenib vehicle and lot are not specified. Exact viability percentages not extracted from bars. Caption asterisks denote p<0.05 versus untreated controls, not a formal interaction test.
Methods and source locations
Compound reported in the paper: PLX4032 (vemurafenib); Compound label in the paper: Vemurafenib (A10739)
Published use explicitly names Adooq A10739; no independent lot identity or purity verification.
Cell viability assay methods; corresponding Figure 6 Results and Figure 6 image/caption; Statistical analysis
MTT cell viability relative to control: Figure 6; mean±SD, three independent experiments. No numeric bar-height estimates or synergy inference. — Cell viability assay methods; corresponding Figure 6 Results and Figure 6 image/caption; Statistical analysis
Paradoxical ERK activation in BRAF wild-type melanoma cells
Cell-based study · BRAF wild-type YUKIM, YUDOSO and YUFIC compared with BRAF V600-mutant YULAC, YURIF, YUMAC and YUGEN8 melanoma strains [3,4]
PLX4032 increased ERK/MEK phosphorylation in the tested BRAF wild-type strains while decreasing it in the V600-mutant strains. Corrected genotype labels are used: YULAC is BRAF V600K and YUKIM carries NRAS Q61R. This signaling result does not establish a universal proliferative response.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 µM PLX4032
- Exposure duration
- 4 and 8 h; DMSO control shown at 4 h
- Method
- Immunoblots for pERK/ERK and pMEK/MEK with actin loading control.
- Vehicle
- 0.1% DMSO control
- Reported limits
- Figure 2A does not provide a biological replicate count or numerical uncertainty for phosphorylation. No numerical fold changes inferred.
Methods and source locations
Compound reported in the paper: PLX4032 (vemurafenib); Compound label in the paper: PLX4032
Literature evidence for the named compound; tested material does not validate an Adooq lot.
Figure 2A image/caption; PLX4032 activates ERK in BRAFWT melanoma cells Results; Western blotting methods; 2012 Corrigendum (DOI 10.1111/j.1755-148X.2012.00995.x); Corrigendum substantive paragraph (retained Wiley extraction L65): YULAC BRAF V600K and YUKIM NRAS Q61R corrections explicitly include Figure 2A; parent identified at L20–35 and L72.
ERK1/2 and MEK phosphorylation: Figure 2A, 1 µM at 4 and 8 h; actin and total ERK/MEK controls. Apply corrigendum to mislabeled genotypes. — Figure 2A image/caption; PLX4032 activates ERK in BRAFWT melanoma cells Results; Western blotting methods; 2012 Corrigendum (DOI 10.1111/j.1755-148X.2012.00995.x); Corrigendum substantive paragraph (retained Wiley extraction L65): YULAC BRAF V600K and YUKIM NRAS Q61R corrections explicitly include Figure 2A; parent identified at L20–35 and L72.
