Buthionine sulphoximine bso

Hoechst 33342 (H3570, 1:500), 2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA. C6827, 1:1000), ThiolTracker™ violet (T10095, 1:500), Alexa Fluor® 633 Phalloidin (A22284, 1:100), Goat anti-mouse Alexa Fluor® 488 (A11001, 1:500), Goat anti-rabbit Alexa Fluor® 633 (A21070, 1:500), Goat anti-mouse Alexa Fluor® 633 (A21050, 1:500) and Goat anti-rabbit Alexa Fluor® 488 (A11008, 1:500) were purchased from Molecular Probes (Invitrogen). Rabbit monoclonal anti-CD44 (EPR10133Y clone, ab51037, 1:1000) rabbit polyclonal anti-EpCAM (ab71916, 1:1000) and mouse monoclonal anti-CD90 (ab133350, 1:1000) were purchased from Abcam (CSP, Cambridge, England). Methotrexate, Doxorubicin, Cisplatin, Sorafenib, Sulfasalazine (SASP), Buthionine sulphoximine (BSO), Arsenic trioxide, Etoposide and Hydrogen peroxide (H₂O₂) were purchased from Sigma Chemicals. Mouse monoclonal anti-CD133/1 (AC133, 130-090-422, 1:100) was purchased from Miltenyi biotec (Bergisch Gladbach, Germany). Rabbit polyclonal anti-AFP (Dako, Denmark A/S, Denmark, A000829. 1:500) and mouse monoclonal anti-β-actin (Sigma, A5441, 1:10,000) antibodies were purchased from each of the indicated companies.

HEK293T cells were cultured in DMEM medium (Thermo Fisher, Waltham, MA, USA) containing 10% heat inactivated fetal bovine serum (FBS) (Thermo Fisher, Waltham, MA, USA), 1% penicillin-streptomycin (Sigma, St. Louis, MO, USA), 1% L-glutamine (Thermo Fisher, Waltham, MA, USA), and 2‰ fungizone (Thermo Fisher, Waltham, MA, USA). Cells were plated at 2.7 × 10⁵ cells/mL density in 8 well ibidi-slide (ibidi, Planegg, Germany). Cell were incubated for 4 h in medium with or without 10 mM buthionine sulphoximine (BSO, Sigma, St. Louis, MO, USA). Cells were incubated for 15 min in PBS containing 50 µM sensor 1, then washed 3 times using warm PBS saline. Plates were exposured under a UV lamp for 10 min to activate 1 prior to imaging. An Olympus FluroView V10i confocal microscope was used for all the imaging. For the imaging of cells treated with GSH sensor, λ_Abs/λ_Em = 559/570–670 nm was used. Same laser intensities and reading sensitivities were used for all imaging sessions. Cell images were processed and the overall fluorescence intensity of each sample was measured using ImageJ Fiji.

Arabidopsis thaliana seeds of wild-type (WT; Col-0), pse1-1 (SALK_046412; Alonso et al., 2003 (link)), and transgenic plants were surface-sterilized and then germinated on half-strength Murashige and Skoog (1/2 MS) medium containing 1% (w/v) agar and 2% (w/v) sucrose at pH 5.8. The seeds were vernalized at 4 °C for 3 d in the dark. The plants were grown in a controlled culture room at 22 °C under long-day (16h of light/8h of dark) conditions with a light intensity of 100 µmol m⁻² s⁻¹.
For the Pb tolerance test, seeds of the WT, the pse1-1 mutant, and the transgenic plants were germinated and grown vertically on 1/2 MS medium in the absence or presence of the indicated concentrations of Pb(NO₃)₂ or the glutathione synthesis inhibitor, buthioninesulphoximine (BSO; Sigma) for 2 weeks, to check whether Pb tolerance in PSE1-overexpressing plants is glutathione dependent. Then, the plants were weighed and their root lengths were measured. There were triplicate replicates for the Pb tolerance test, and ~30 plants were used for each measurement. For Pb-inducible gene expression analysis, the plants were grown for 2 weeks on 1/2 MS medium and then exposed to a solution of 0.5mM Pb(NO₃)₂ or water (control) at the designated time points specified in the text.