# Methylene blue molecular-target map

Scope: selected experimental anchors, compiled 12 September 2026. This document distinguishes the measured response from a proposed direct target. Experimental concentrations describe the cited assay, not administration instructions.

| Process | Model | Evidence type | Experimental anchor | Endpoint | Boundary | Primary study |
| --- | --- | --- | --- | --- | --- | --- |
| MAO-A / MAO-B | Purified recombinant human enzymes | Biochemical kinetics | MAO-A tight-binding analysis; Ki 27 ± 3 nM | Enzyme activity | Ki differs from IC50; neither specifies tissue exposure | [Ramsay 2007](https://pubmed.ncbi.nlm.nih.gov/17721552/) |
| NOS / soluble guanylyl cyclase | Purified enzyme, endothelial cells, isolated vessels | Biochemical and functional | Complete NOS inhibition at 30 μM; approximately 50% stimulated cyclase inhibition at 60 μM | Citrulline, cGMP, vessel relaxation | Decreased cGMP does not prove selective cyclase inhibition | [Mayer 1993](https://pubmed.ncbi.nlm.nih.gov/7679577/) |
| Electron transport | Transformed mouse hippocampal HT-22 cells and biochemical reactions | Cellular bioenergetics and redox assays | Oligomycin, FCCP, rotenone comparator sequence | Oxygen consumption, acidification, ATP | Oxygen consumption and ATP were separate assays | [Wen 2011](https://pmc.ncbi.nlm.nih.gov/articles/PMC3091255/) |
| Complex III bypass | Isolated mouse brain mitochondria | Organelle inhibitor experiment | 1 μM MB with rotenone or antimycin; pyruvate/malate-supported respiration | Respiration, membrane potential | Partial complex I rescue did not extend to antimycin-blocked complex III | [Complex III study](https://pmc.ncbi.nlm.nih.gov/articles/PMC6433145/) |
| Nrf2 | Wild-type and Nrf2-knockout mouse embryonic fibroblasts | Genetic dependency | 0.1, 1, 10 μM MB; vehicle comparator | HO1, NQO1, Gclc transcripts | Dependence on Nrf2 does not establish direct MB binding | [Stack 2014](https://pmc.ncbi.nlm.nih.gov/articles/PMC4065148/) |
| Autophagy | CHO cells expressing human wild-type 4R0N tau | Reporter plus genetic perturbation | 0.01 μM MB, 6 h; vehicle, bafilomycin, BECN1 knockdown comparisons | Tandem LC3 reporter, tau, signaling proteins | Vesicle abundance differs from flux; gene knockdown adds causal evidence | [Congdon 2012](https://pmc.ncbi.nlm.nih.gov/articles/PMC3405840/) |
| Hsp70 / tau disposal | Purified Hsp70 and tau-expressing HeLa cells | Biochemical plus cellular | ATPase screening and proteasome-dependence experiments | ATPase activity and tau abundance | Proteasomal disposal differs from lysosomal autophagy | [Jinwal 2009](https://pubmed.ncbi.nlm.nih.gov/19793966/) |
| Tau condensates | Reconstituted recombinant-tau droplets; separate SH-SY5Y cytotoxicity assay | Biophysical experiments plus a separate cellular viability endpoint | Phase-separation and gelation experiments; SH-SY5Y exposure to incubated tau species for 24 h | Droplet formation and material state in vitro; MTT viability in cells | Cellular viability does not establish intracellular condensate formation | [Nature Communications 2023](https://www.nature.com/articles/s41467-023-41241-6) |

## Reading sequence

1. Identify the material and experimental system.
2. Record the measured endpoint, concentration, duration, and comparator.
3. Separate direct biochemical action, pathway dependence, and organism phenotype.
4. Check whether an independent assay supports the same interpretation.
5. Preserve contrary findings and model boundaries in any summary.

Definitions: MB, methylene blue; MAO, monoamine oxidase; NOS, nitric oxide synthase; CHO, Chinese hamster ovary; ATP, adenosine triphosphate; Ki, inhibition constant; IC50, concentration giving half-inhibition under specified assay conditions.