Azd8055

The ERK inhibitor SCH772984 was purchased from SelleckChem (S7101), Z-Leu-Leu-Leu-al (MG132, S2619) were purchased from Fisher and mTOR inhibitor AZD8055 (HY_10422) MedChem Express.

All temporal chemical treatments were performed using 10 day old seedlings. Each of the following chemicals was first dissolved in DMSO to prepare a stock solution, and then diluted in Arabidopsis growth medium (½ LS and 1% sucrose) to reach the specific working concentration. 33 μM Wortmannin (Sigma-Aldrich, W1628) and 100 μM LY294002 (MedChemExpress, HY-10108) were used to treat seedlings for 2 hr. Latrunculin B (Sigma-Aldrich, L5288) and Oryzalin (Chem Service Inc, N-12729) were diluted to 25 μM and 40 μM, respectively, for 2 hr treatments. For TOR inhibition, seedlings were incubated with 5 μM AZD-8055 (MedChemExpress, HY-10422) or 1 μM Torin2 (MedChemExpress, HY-13002) for 2 or 4 hr as the figure legends indicated. 10 μM solution of β-estradiol (Sigma-Aldrich, E8875) was used to induce gene silencing.

After seven days of growth (in the middle of logarithmic phase), cells were centrifuged at 600× g for 5 min and resuspended in OHM without additives (control), or in OHM containing NaCl at a final concentration of 0.2% (w/v) or 0.8% (w/v), respectively, or in OHM containing 0.2% or 0.8% of NaCl supplemented with AZD8055 ((5-(2,4-bis((S)-3-methylmorpholino)pyrido[2,3-d]pyrimidin-7-yl)-2-methoxyphenyl)methanol; MedChemExpress) at a final concentration of 0.2 µM. Cell density at the beginning of experiments was 50,000–150,000 cell per ml culture depending on the experiment. The cultures were then grown as described above. Cells were collected for analyses after 1, 3, and 5 days of cultivation, respectively.

Trastuzumab (Roche Pharmaceutical Ltd., Switzerland) was solubilized in sterile water containing 1.1% benzyl alcohol (stock solution at 100μg/mL, stored at 4°C). Chemicals used are as follows: AZD8055 (Medchem express, USA), dimethyl sulfoxide (DMSO) (Amresco, USA), Dithiothreitol (DTT) and iodoacetamide (IAA) (Sigma-Aldrich, USA), DAPI (Beyotime Biotechnology, China). Cell Count kit-8 was purchased from Dojindo (Kumamoto, Japan). Antibodies against the following proteins were used for immunoblotting: HER-2 (Abcam, UK), mTOR (Abcam, UK), AKT1S1 (Abcam, UK), RPS6KB1 (Cell Signaling Technology, USA), p-RPS6KB1 ^{(Thr421/Ser424)} (Cell Signaling Technology, USA), AKT (Cell Signaling Technology, USA), p-AKT^(Ser473) (Cell Signaling Technology, USA), β-actin (Cell Signaling Technology, USA). Horseradish peroxidase-labeled goat anti-rabbit and anti-mouse secondary antibodies were purchased from ZSGB-BIO (Beijing, China).

Murine IL-3 was purchased from Sigma-Aldrich. AZD6244, MEK162 and PD0325901 were purchased from Selleck Chemical. Everolimus and AZD8055 were purchased from MedChemExpress. All inhibitors were solubilized in dimethyl sulfoxide (DMSO) at stock concentrations of 1 mM.

AZD6244, MEK162, BKM120 and PD0325901 were purchased from Selleck Chemical. Everolimus, AZD8055, GSK690693, MK2206 and BKM120 (as a control) were purchased from MedChemExpress. All inhibitors were solubilized in dimethyl sulfoxide (DMSO) at stock concentrations of 1mM.

The xenograft (P1) was dissociated into individual cells, which were subsequently cultured in DMEM medium at 37 °C with 5% CO2. The culture medium consisted of 10% FBS and 1% P/S. Passaging was performed every 3–4 days, and during this process, cells were evenly preserved by freezing them in a medium containing 10% DMSO, either in liquid nitrogen or at -80 °C for future use [29 (link)]. It is noteworthy that cells within 5 generations were exclusively utilized to prevent aging. The PI3K inhibitors, BEZ235 (MedChemExpress, HY-50,673) and AZD8055 (MedChemExpress, HY-10,422), were dissolved in DMSO. Subsequently, the cells were uniformly seeded in a 24-well plate at a density of 5 × 104 cells per well, and they were cultured under suitable conditions with the addition of growth medium. The final concentration of BEZ235 was set at 100 nM, and cells were harvested after 72 h of treatment [28 (link)]. For the AZD8055 treatment group, the final concentration was 50 nM, and cells were collected after 48 h of treatment [29 (link)]. Protein extraction was performed for subsequent analyses. The specific experimental groups encompassed the control group (con group), the BEZ235 group, and the AZD8055 group.