In CFBE41o− cells expressing F508del-CFTR, Spautin-1 (10 µM, final 3 h) reduced the rescue produced by VX-809 (1 µM, 24 h) by nearly 40% in a functional assay and lowered CFTR protein abundance. SAR405 and torin-1 did not reproduce this CFTR phenotype despite evidence of autophagy modulation, cautioning against attributing the effect solely to autophagy inhibition. [2]
Inhibition of USP10 and USP13 deubiquitinating activity
Biochemical assay · USP10 and USP13 isolated from 293T cells; Ub-AMC and ubiquitinated Beclin1 substrates; CYLD comparator [1]
Spautin-1 inhibited USP10 and USP13 in a fluorogenic substrate assay. Beclin1-substrate assays supported inhibition, with a catalytically inactive USP10 control. CYLD was not inhibited in the Ub-AMC comparison; this limited comparator does not establish proteome-wide selectivity.
Literature-Reported Treatment Conditions
- Treatment concentration
- 100 nM–20 µM in Ub-AMC assay; 10 and 20 µM in Beclin1-substrate panels
- Exposure duration
- Ub-AMC: 30 min preincubation, then 45 min with substrate; Beclin1 substrate: 2 h
- Method
- Cell-free deubiquitination assays
- Reported limits
- Replicate number and biochemical assay solvent concentration are not specified in the adopted main-text caption/methods.
Methods and source locations
Compound reported in the paper: Spautin-1; Compound label in the paper: C43
Research evidence for the named compound; no salt conversion, clinical efficacy or validation of an Adooq lot is inferred.
Fig 1D and identity Results; Fig 4F–I/captions and DUB Results; Experimental Procedures
Inhibition of USP10 and USP13 Ub-AMC hydrolysis: Fluorogenic Ub-AMC hydrolysis; final protein 20 nM and substrate 0.8 µM; error bars STD. Figure labels give USP10 0.5779 µM and USP13 0.6916 µM; the adopted range is the authors’ rounded Results statement, not digitization. — Fig 4H–I and DUB-identification Results
Deubiquitination of ubiquitinated Beclin1: In vitro immunoblot substrate assay with USP10/USP13 and USP10CA control — Fig 4F–G, captions; In Vitro Deubiquitination Assay
Cellular degradation of Beclin1/Vps34 complexes
Cell-based study · H4-LC3-GFP cells; 293T expression/immunoprecipitation controls [1]
Spautin-1 lowered endogenous Beclin1, Vps34 and associated complex proteins along with LC3-II. Cycloheximide and degradation-inhibitor controls supported protein degradation rather than a simple synthesis effect. MG132, unlike NH4Cl, opposed the loss of Beclin1/Vps34. Beclin1 ubiquitination increased in the expression-system assay.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 µM for time course, inhibitor-rescue and ubiquitination experiments; 1–10 µM in CHX comparison
- Exposure duration
- Up to 12 h (time course); 12 h (CHX); 6 h (degradation inhibitors); 24 h (ubiquitination)
- Method
- Cell treatment followed by immunoblot or immunoprecipitation
- Vehicle
- DMSO 1‰ for Fig 3G–H; caption specifies vehicle treatment
- Co-treatment
- CHX, MG132 or NH4Cl in mechanistic controls; MG132 5 µM for the last 6 h of the 24 h ubiquitination experiment
- Reported limits
- Main caption does not specify independent replicate counts for these immunoblots.
Methods and source locations
Compound reported in the paper: Spautin-1; Compound label in the paper: C43
Research evidence for the named compound; no salt conversion, clinical efficacy or validation of an Adooq lot is inferred.
Fig 3F–I/captions, Vps34-complex-degradation Results; cell culture/antibody Methods
Beclin1/Vps34-complex abundance and LC3-II: Immunoblot with β-tubulin loading control; 0, 2, 4, 8, 10 and 12 h shown — Fig 3F/caption and Vps34-complex-degradation Results
Loss of Beclin1/Vps34 with synthesis or degradation inhibitors: CHX 10 µM with Spautin-1 1, 5 or 10 µM for 12 h; MG132 10 µM or NH4Cl 10 mM with Spautin-1 10 µM for 6 h; β-tubulin control — Fig 3G–H/captions
Ubiquitinated Beclin1: 293T GFP-Beclin1/HA-Ub transfection; GFP immunoprecipitation and anti-HA immunoblot; input/global ubiquitination comparison — Fig 3I/caption
Antagonism of VX-809 rescue of F508del-CFTR
Cell-based study · CFBE41o− cells stably expressing F508del-CFTR and halide-sensitive YFP [2]
Spautin-1 reduced the VX-809-associated functional rescue and lowered mature band C and total CFTR abundance. These are combination-context effects on mutant CFTR, not a biochemical IC50 or evidence of an anticancer effect.
Literature-Reported Treatment Conditions
- Treatment concentration
- Spautin-1 10 µM; VX-809 1 µM
- Exposure duration
- VX-809 or vehicle for 24 h; Spautin-1 or vehicle during final 3 h
- Method
- Cell pretreatment, HS-YFP functional assay and immunoblot
- Vehicle
- DMSO
- Co-treatment
- VX-809 correction; forskolin 20 µM plus genistein 50 µM used for functional assay stimulation
- Reported limits
- Supplier, salt form and final DMSO percentage are not specified in the retrieved main text.
Methods and source locations
Compound reported in the paper: Spautin-1; Compound label in the paper: Spautin-1
Research evidence for the named compound; no salt conversion, clinical efficacy or validation of an Adooq lot is inferred.
Fig 1A–B/caption and opening Results; fluorescence-assay, Western Blot and Statistics Methods
VX-809-induced F508del-CFTR functional rescue: HS-YFP iodide-quenching assay; n = 14–23 independent experiments; mean and SEM; Fig 1A marks p < 0.001. Approximation is reported in Results, not estimated from the bars. — Results opening paragraph; Fig 1A/caption; fluorescence-assay and Statistics Methods
Mature CFTR band C and total bands C+B: Immunoblot normalized to GAPDH; n = 3 independent experiments; mean and SEM; caption p < 0.05 — Fig 1B/caption; opening Results; Western Blot and Statistics Methods
Autophagy-comparator controls for the CFTR phenotype
Cell-based study · CFBE41o− cells expressing F508del-CFTR; functional, immunoblot and MDC readouts [2]
SAR405 and torin-1 did not reproduce a change in VX-809-rescued CFTR function. SAR405 also did not reproduce the loss of CFTR protein seen with Spautin-1. LC3-II and MDC controls demonstrated activity of the autophagy modulators in these cells. These pharmacologic comparisons caution against assigning the CFTR phenotype solely to autophagy inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- VX-809 1 µM; SAR405 0.01–10 µM in Fig 8A; torin-1 0.01–25 nM in Fig 9A
- Exposure duration
- VX-809 24 h and Spautin-1/SAR405 3 h in Fig 8B; MDC method describes test-compound exposure for 24 h
- Method
- Comparative pharmacology with functional and autophagy-marker controls
- Vehicle
- DMSO is the negative control in MDC Methods; vehicle comparator in functional captions
- Co-treatment
- Parallel comparator conditions, not a therapeutic combination. Torin-1 with Spautin-1 or SAR405 and bafilomycin A1 in Fig 9C.
- Reported limits
- Exact Spautin-1/SAR405/torin-1 concentrations and timing for every marker panel are not specified in the retrieved main captions; no conditions are borrowed from Fig 1. Functional Fig 8A and 9A exposure duration is not specified in those captions.
Methods and source locations
Compound reported in the paper: Spautin-1; Compound label in the paper: Spautin-1
Research evidence for the named compound; no salt conversion, clinical efficacy or validation of an Adooq lot is inferred.
Figs 8–10/captions, associated Results; MDC, Western Blot and Statistics Methods
VX-809-rescued F508del-CFTR function and protein abundance: HS-YFP: SAR405 0.01–10 µM (Fig 8A), torin-1 0.01–25 nM (Fig 9A); immunoblot of CFTR/GAPDH (Fig 8B, n = 3 independent experiments); Fig 9A n = 3 separate experiments — Results autophagy-comparator paragraphs; Figs 8A–B and 9A/captions
Autophagy-associated LC3-II and MDC signal: LC3-II/GAPDH immunoblot with bafilomycin A1 controls (Fig 9B–C); MDC imaging (Fig 10); n = 3 separate experiments in each caption; mean and SEM — Figs 9B–C and 10/captions; associated Results; MDC, Western Blot and Statistics Methods
