RSL3 responses depend on the cellular model. In a stereoisomer comparison, (1S,3R)-RSL3 was more potent than (1R,3R)-RSL3 in HT1080 cells, whereas both produced similar 72-hour growth inhibition in colorectal cancer cells. Growth inhibition alone does not establish ferroptosis. [3]
GPX4 identification with an RSL3-derived affinity probe
Biochemical assay · BJeLR cells; affinity enrichment and quantitative proteomics [1]
An active (1S,3R)-RSL3 fluorescein probe enriched GPX4 relative to an inactive stereoisomer probe and free-parent competition; immunoblotting confirmed enrichment. The tagged probe is chemically distinct from free RSL3.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.5 μM active or inactive fluorescein probe; competitor arm: 0.5 μM free (1S,3R)-RSL3
- Exposure duration
- Probe exposure 2 h; free-parent competitor pretreatment 20 min
- Method
- Intact-cell probe treatment followed by affinity purification
- Vehicle
- Matched vehicle pretreatment; formulation not specified in the read main method
- Co-treatment
- Free-parent competition control; inactive (1R,3R)-probe control
- Reported limits
- Full SI target-analysis details not retrieved; tagged probe potency is not parent potency.
Methods and source locations
Compound reported in the paper: RSL3; Compound label in the paper: (1S,3R)-RSL3 parent competitor; (1S,3R)-RSL3-fluorescein active probe
Evidence for the reported compound and model; no validation of an Adooq lot.
Fig. 3C, E–H; Results RSL3 Binds to and Inactivates GPX4; Methods RSL3 Target Identification
GPX4 enrichment with active affinity probe: Affinity purification, label-free LC-MS/MS and GPX4 immunoblot; three independent affinity preparations per treatment, each subsampled in three LC-MS/MS analyses. — Fig. 3E–H; Results target-identification paragraphs; Methods RSL3 Target Identification
Purified-enzyme profiling limits a direct GPX4-inhibition interpretation
Biochemical assay · Recombinant human selenoprotein GPX4 and TXNRD1; GPX1 and glutathione reductase comparators [2]
RSL3 did not inhibit purified GPX4 up to 80 μM in the reported assay, while inhibiting TXNRD1. This biochemical result limits interpretation of RSL3 as a selective direct GPX4 inhibitor; it does not invalidate cellular GPX4-associated effects.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response profiling; GPX4 tested up to 80 μM
- Exposure duration
- 30 min compound preincubation in the main biochemical protocol
- Method
- Cell-free enzyme assays
- Vehicle
- DMSO
- Reported limits
- No single assay-independent IC50 assigned. Supplementary and main assays give different TXNRD1 potency values. Supplier/lot identity is not documented in the read text.
Methods and source locations
Compound reported in the paper: RSL3; Compound label in the paper: (1S,3R)-RSL3
Evidence for the reported compound and model; no validation of an Adooq lot.
Results 2.1 and Discussion; Fig. 1A; Methods Biochemical assays; Supplementary Results p. 2 and Extended Data Figs. 1–2
Purified GPX4 activity: Glutathione-reductase-coupled NADPH-consumption assay with cumene hydroperoxide; compound preincubation 30 min; DMSO control. Extended Data Fig. 2 caption: n = 3 technical replicates. — Results 2.1; Methods Biochemical assays; Supplementary Results p. 2; Extended Data Fig. 2A
Purified TXNRD1 activity: Selenite-reduction and DTNB assays; DMSO normalization; oxidized versus NADPH-reduced enzyme comparison in Extended Data Fig. 2B. — Fig. 1A; Results 2.1; Supplementary Results p. 2 and Extended Data Fig. 2A–B
Model-dependent stereoisomer effects on cell growth
Cell-based study · HT1080 fibrosarcoma and DLD1, HCT116, RKO and SW480 colorectal cancer cell lines [3]
At 72 h, (S)-RSL3 was more potent than (R)-RSL3 in HT1080 cells, but the two stereoisomers were nearly equipotent in the colorectal cell panel. These are cell-growth EC50 values, not biochemical IC50 values or proof that every growth effect is ferroptosis.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response; HT1080 panel: 0.0078–2 μM; CRC panel plotted across approximately 0.01–10 μM
- Exposure duration
- 72 h
- Method
- Label-free live-cell imaging with Cytation 5/BioSpa
- Reported limits
- Vehicle composition for these growth experiments not established; replicate unit described as wells. Rescue-agent unit inconsistencies in unselected panels are unresolved.
Methods and source locations
Compound reported in the paper: RSL3; Compound label in the paper: (S)-RSL3 = (1S,3R)-RSL3; (R)-RSL3 = (1R,3R)-RSL3 comparator
Evidence for the reported compound and model; no validation of an Adooq lot.
Fig. 1B, D–F; Results The potency of (S)-RSL3 is due to GPX4 independent effects in CRC cell lines; Methods Cell lines and Growth assays
HT1080 adherent cell-growth inhibition: 72 h label-free live-cell imaging; relative cell-number fold change normalized to control. Figure table gives 13.24 nM and 903.1 nM; rounded prose values retained. — Fig. 1D–E and Results first section; Methods Growth assays
Colorectal cell-growth inhibition: 72 h live-cell imaging, normalized growth. Main Results reports DLD1 about two-fold more sensitive to (R)-RSL3. General method specifies at least three independent wells per condition and mean ± SD. — Fig. 1F and Results first section; Methods Growth assays
