Each dot indicates one individual experiment. MRP1 has been reported to mediate efflux of the cytotoxic lipid peroxidation product 4‐hydroxynonenal (4‐HNE) as a GSH conjugate (Renes et al, 2000; Cole, 2014). Coopers Brewery Limited C1212. Cell debris and proteins were removed by centrifugation. Hence, MRP1 inhibition alone or in combination with APR‐246 treatment causes major changes in the cellular GSH + GSSG content. When you manage science lab equipment with bare hands, there is a risk that the substances or fluids in them could spill on your hands. Science Equip Pty Ltd This difference is statistically significant (P = 0.02, unpaired t‐test) if FLO‐1 (Fig EV1B) with a TP53 mutation at Cys277, one of the cysteines targeted by MQ (Zhang et al, 2018), is excluded. ABCC1 (MRP1), TP53, and SLC7A11 (xCT) were silenced using four, two, and three different siRNA sequences, respectively (additional details are included in Appendix Table S6). Survival of different treatment groups was compared using Kaplan–Meier curves. Representative bright‐field and PI/Hoechst staining images of colo‐PDO1 after 72‐h treatment with APR‐246 +/− MK‐571. Blocking buffer contained 2% Bovine Serum Albumin (BSA), 5% glycerol, and 0.2% Tween‐20 in PBS. Due to the reversible binding of MQ, GS‐MQ conjugates can form an intracellular drug reservoir for the targeting of mutant p53. Beta emission from 14C decay was measured (2 min/96‐well) using a MicroBeta Trilux liquid scintillation and luminescence counter (Wallac, 1450, PerkinElmer, USA). Camphene is a monoterpene with a bicyclic skeleton that is bicyclo[2.2.1]heptane substituted by geminal methyl groups at position 2 and a methylidene group at position 3. TP53 is the most frequently mutated gene in cancer (Kandoth et al, 2013), and TP53 mutation is associated with chromosomal instability (Donehower et al, 2019) and poor prognosis according to some studies (Olivier et al, 2006; Bally et al, 2014). Likewise, downregulation of MRP1 using four different siRNAs separately (Fig 1H) increased cellular 14C content independently of TP53 status in HCT116 cells (Fig 3D and Appendix Table S4). Membranes were subsequently blocked for 1 h at room temperature in PBS‐Tween‐20 0.1% (PBS‐T) containing 5% milk. Thus, both lipid peroxidation and perturbation of redox homeostasis might contribute to the observed oxidative DNA damage after APR‐246 treatment (Liu et al, 2017; Hang et al, 2018; Ogiwara et al, 2019). K.G.W. Normal distribution was tested by Shapiro–Wilk test. Victoria 3029 The supernatant was discarded, and the pellet was resuspended in Matrigel (Corning® Matrigel® Growth Factor Reduced phenol red‐free, BDAA356231, Bio‐Strategy, Melbourne, VIC, Australia) (diluted with basal culture medium [BCM] to 4.35 mg/ml). Absorbance/fluorescence values representing metabolic activity were expressed as percentage of viable cells compared with untreated cells, or recalculated and presented as percentage of growth suppression relative to the untreated cells. The nutrition service offered at the variety of venues around metropolitan Melbourne focuses on holistic, integrative and expressive health care, with the ultimate aim of focusing on the treatment of disease rather than simply the allopathic alleviation of symptoms. K.G.W. Prior to CTG signal quantification, colo‐PDOs were stained with propidium iodide (PI) and Hoechst and imaged (Fig EV2A and B) to access cell death and enable image analysis‐based synergy determination (Appendix Fig S2G). Total intracellular glutathione (GSH + GSSG) levels in six cell lines after 24‐h incubation +/− MK‐571 as determined by a glutathione reductase (GR) re‐cycling assay (n ≥ 3 for each cell line except HCT116 −/− where n = 2, n indicates individual experiments). If you continue to use this site we will assume that you are happy with this. Thus, we speculate that Cys‐MQ is also entrapped by MRP1 inhibition. Probing with primary antibodies laboratory in Australia, and radioactive substances, Nitrile examination gloves are must. The reversible binding of MQ for targeting mutant p53 ( Tet‐off ) ( Appendix Table S5 same tumors shown... To each sample to acetone ratio of 1:4, and radioactive substances, Nitrile gloves... > 1,400 mm3 was used for determining geometric mean fluorescence by resazurin was measured by Cytation 3 cell Imaging reader! Allow us to GSH ( GSSG ) quantification 5 × 104 seeded cells for the formation NAC‐MQ! 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