Pi rnase solution

BMDM were treated with Cy5.5-conjugated NPs for 1.5 h then washed twice with PBS. 1% paraformaldehyde in PBS was added to the cells for fixation for 15 min at room temperature. The cells were then washed and simultaneously blocked and permeabilized with a 1% BSA and 0.1% Triton X-100 PBS solution for 30 min at room temperature. Cell were then stained with a LAMP1-eF450 (1:100; Ex/Em: 405 nm/450 nm; eBioscience) primary antibody in PBS for 30 min. BMDM were washed again and immediately prior to mounting were stained with 0.5 mL/million cells of PI-RNAse solution (Ex/Em: 493 nm/636 nm; BD). BMDM were imaged on the Zeiss LSM800 AxioObserver Z1. Images were later analyzed using ImageJ.

We detached 5 × 10⁵ cells from the plates using 3mM EDTA/PBS and resuspended in 500 μl of ice-cold PBS. Annexin V-fluorescein isothiocyanate (FITC) staining was performed according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA). For cell cycle profile analysis, cells were fixed using 80 % ethanol, stored overnight at 4 °C, and then spun at 1,500 rpm for 5 minutes at 4 °C using a centrifuge (Eppendorf 5810R). Pellets were washed with cold PBS + 1 % serum, mixed, spun for 5 minutes at 1,200 rpm, and stained with propium iodide (PI)/RNase solution (BD Pharmingen). All analysis was performed using FACS Diva software on a BD FACSCanto II flow cytometer.

Cells of different groups as previously described were harvested and washed with PBS and fixed with 75% ethanol at 4°C overnight. After being stained with PI/RNase solution (BD Pharmingen, 550825) for 15 min in the dark, cells cycle distribution of different groups was detected by flow cytometry (Becton Dickinson, USA).

Megakaryocyte ploidy was analyzed on day 11 of culture. Cells were labeled with APC-conjugated anti-CD41 antibody, incubated for 20 minutes on ice, fixed with 2% paraformaldehyde (Fischer Scientific) for 15 minutes and then washed with PBS (Cellgro, Manassas, VA). Next, cells were permeabilized with a BSA 0.02% + saponin 0.005% (MP Biomedicals, Solon, OH, USA) and incubated for 15 minutes at room temperature. Finally cells were treated with Propidium Iodide (PI)/RNase solution (BD, Franklin Lakes, NJ) and incubated for 30 minutes in dark at room temperature before flow cytometry analysis.

Hep3B and SK-Hep1 cells (5 × 10⁵) were seeded in 6-well plates and incubated with 50 μM magnolol, 10 μM regorafenib, and a combination for 48 h. ZVAD was added for 30 min before magnolol combined with regorafenib for 48 h. The cells were then harvested for the staining of different reagents, including Annexin-V/PI, cleaved-caspase-3 (1 μL, fluorescein isothiocyanate-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (FITC-DEVD-FMK)), cleaved-PARP-1, FAS-FITC (1 μL), FAS-L-PE (1 μL), cleaved-caspase-8 (1 μL, sulforhodamine-Ile-Glu-Thr-Asp-fluoromethyl ketone (Red-IETDFMK), cleaved-caspase-9 (1 μL FITC-Leu-Glu-His-Asp-fluoromethyl ketone (FITC-LEHD-FMK)), DCFH-DA (500 μL at 10 μM) for ROS, DIOC6 (4 μmol/L) for Dy loss, and Fluo-3/AM (2.5 μg/mL) for Ca²⁺. For subG1 analysis, the harvested cells were fixed by 70% ethanol overnight at −20 °C and stained by a PI/RNase solution (cat: 550625, BD Biosciences). The fluoresce signal from the cells was detected and quantified by NovoCyte flow cytometry and the NovoExpress^® software (Agilent Technologies Inc., Santa Clara, CA, USA) [15 (link),17 (link)].

Cell cycle distribution was analysed by flow cytometry (Becton Dickinson). Cells were synchronized in growth factor-free keratinocyte-SFM with 2 mM thymidine (catalogue no.T1895, Sigma-Aldrich) for 24 h. Transfection is performed using Lipofectamine TM RNAiMAX (Invitrogen) according to its manual, and the final concentration of annealed oligo RNAs is 400 nM. Four hours after transfection, culture medium was replaced by complete keratinocyte-SFM supplemented with L-glutamine, prequalified human recombinant epidermal growth factor (EGF) 1-53 and BPE. Cells were harvested at different time points, washed in PBS, fixed with ice-cold 70% ethanol and strained in PI/RNase solution (BD Pharmingen). The samples were analysed on a FACScan flow cytometer in combination with BD lysis software (Becton Dickinson).

Cells were harvested after exposure to various concentrations of Sme for 48 hours and fixed with 70% ethanol at 4°C overnight. The cells were then stained with PI‐RNAse solution (BD Biosciences) for 30 minutes in the dark. Then the samples were analyzed cell cycle distribution by flow cytometer (BD Biosciences).