In primary rat oligodendroglial cultures, 50–200 nM lactacystin for 72 hr increased morphological complexity and differentiation-associated markers. Responses varied by marker: A2B5-positive cells were unchanged at 50 nM and decreased at 100–200 nM. These findings concern cultured-cell differentiation rather than demonstrated functional myelination. [1]
Lactacystin produced a dose- and time-dependent MTT response in the same cell model. At 72 hr, 100–200 nM increased the signal, whereas concentrations above 200 nM reduced it below vehicle controls. MTT is a metabolic readout, so the result should not be interpreted as a direct count of surviving cells. [1]
In mouse bone marrow-derived macrophages expressing wild-type or D162E MyD88, lactacystin (50 μM, 5 h) did not fully prevent MyD88 degradation. This qualitative result limits a solely proteasomal explanation of turnover, but does not establish complete proteasome inhibition or quantify other degradation pathways. [2]
Proteasome-associated activities after cellular lactacystin exposure
Cell-based study · Primary oligodendroglial cultures from newborn rat cerebral hemispheres; cultures reported as 95% pure. [1]
After 200 nM lactacystin for 72 hr, lysate trypsin-like, chymotrypsin-like and PGh-like activities decreased versus matched vehicle controls. These are cellular-exposure results, not a purified-enzyme IC50.
Literature-Reported Treatment Conditions
- Treatment concentration
- 200 nM
- Exposure duration
- 72 hr
- Method
- Cell treatment followed by fluorogenic substrate assays in lysates
- Vehicle
- DMSO, 1 × 10^-3%
- Co-treatment
- No cotreatment; MG132 was evaluated separately.
- Reported limits
- The assay methods also describe measurements with and without 50 μM lactacystin; this assay condition is distinct from the 200 nM culture exposure. No IC50 was determined in this experiment.
Methods and source locations
Compound reported in the paper: Lactacystin; Compound label in the paper: lactacystin
Partial suppression of measured proteolytic activities in rat oligodendroglial cultures.
Figure 5, embedded table and caption; Results: Proteolytic activities of the proteasome; Materials and Methods: Oligodendroglial cell primary cultures; Proteolytic activities of the 26S proteasome; Statistical analysis
Trypsin-like activity decrease relative to control: Lysate fluorogenic Boc-Leu-Ser-Thr-Arg substrate assay — Figure 5 embedded table and caption; Results: Proteolytic activities of the proteasome
Chymotrypsin-like activity decrease relative to control: Lysate fluorogenic Ala-Ala-Phe substrate assay — Figure 5 embedded table and caption; Results: Proteolytic activities of the proteasome
PGh-like activity decrease relative to control: Lysate Clz-Leu-Leu-Glu-β-naphthylamide substrate assay — Figure 5 embedded table and caption; Results: Proteolytic activities of the proteasome
Morphological and marker changes in oligodendroglial cultures
Cell-based study · Primary oligodendroglial cultures from newborn rat cerebral hemispheres; cultures reported as 95% pure. [1]
A 72 hr lactacystin exposure increased morphological complexity and differentiation-associated markers. A2B5-positive cells decreased at 100–200 nM; the 50 nM condition did not significantly alter this marker.
Literature-Reported Treatment Conditions
- Treatment concentration
- 50, 100 or 200 nM
- Exposure duration
- 72 hr
- Method
- O4-positive morphological scoring and immunocytochemistry
- Vehicle
- DMSO, 1 × 10^-3%
- Co-treatment
- No cotreatment; MG132 comparison was separate.
- Reported limits
- Individual-cell counts per condition are not specified in the adopted figure captions. Morphology Figure 2 reports three independent experiments; do not transfer that n to other panels.
Methods and source locations
Compound reported in the paper: Lactacystin; Compound label in the paper: lactacystin
Differentiation-associated morphology and marker expression in cultured cells, without inference of functional myelination in animals.
Figures 2 and 4 and captions; Results: morphological complexity and marker evaluation; Methods: primary cultures, morphological analysis, immunocytochemistry
Morphology and differentiation-associated immunoreactivity: Blind morphological scoring and marker-positive cell quantification — Figures 2 and 4; corresponding Results and captions
Dose- and time-dependent MTT response
Cell-based study · Primary oligodendroglial cultures from newborn rat cerebral hemispheres; cultures reported as 95% pure. [1]
The MTT response depended on concentration and time. At 72 hr, 100–200 nM increased signal while concentrations above 200 nM reduced it below vehicle control. The lower signal at higher exposure limits interpretation of differentiation findings.
Literature-Reported Treatment Conditions
- Treatment concentration
- 25–5000 nM, as plotted in Figure 1
- Exposure duration
- 24 or 72 hr
- Method
- MTT absorbance at 570 nm
- Vehicle
- DMSO, 1 × 10^-3%
- Co-treatment
- No cotreatment.
- Reported limits
- Methods specify six samples per condition, without identifying these as six independent biological experiments. Exact absorbance values were not tabulated in the adopted text and were not estimated from bars.
Methods and source locations
Compound reported in the paper: Lactacystin; Compound label in the paper: lactacystin
MTT metabolic viability readout; no inference of an IC50, direct cell count or universal prosurvival effect.
Figure 1A–B and caption; Results: Effects of lactacystin on OLGc viability; Methods: MTT assay, primary cultures and statistical analysis
MTT signal relative to vehicle across dose/time: MTT assay, six samples per experimental condition; biological replicate number not assigned — Figure 1A–B; Results: Effects of lactacystin on OLGc viability; Methods: MTT assay
Incomplete prevention of MyD88 degradation in macrophages
Cell-based study · Mouse bone marrow-derived macrophages expressing MyD88wt or MyD88D162E [2]
Lactacystin did not fully prevent MyD88 degradation during the 5 h experiment. The authors therefore considered a contribution from degradation routes beyond the proteasome. The experiment does not quantify the fraction attributable to each pathway.
Literature-Reported Treatment Conditions
- Treatment concentration
- 50 μM
- Exposure duration
- 5 h
- Method
- Cell exposure and MyD88 Western blot with β-actin loading control
- Co-treatment
- Cycloheximide (100 μM) was a separate comparison condition, not a lactacystin combination.
- Reported limits
- Lactacystin vehicle, CAS, catalog number, lot, purity and stereochemical verification were not specified in the read treatment methods. No quantitative rescue percentage was reported for panel B.
Methods and source locations
Compound reported in the paper: Lactacystin; Compound label in the paper: lactacystin
A qualitative, incomplete proteasome-inhibitor rescue in the stated macrophage genotypes.
Figure 2B and caption; Results paragraph beginning “To further characterize”; Methods: Western blot
MyD88 protein after inhibition of proteasomal degradation: MyD88 immunoblot; β-actin loading control; representative of two independent experiments — Figure 2B and caption; first BMDM Results paragraph
