Cck8 kit

According to the standard protocol, Th22 cells were seeded into a 96-well plate at a density of 5000 cells/well and cell proliferation was measured using the CCK8 Kit (7Sea Biotech, Shanghai, China) according to the manufacturer’s instructions. Cell viability was evaluated using the WST-1 assay (Roche Diagnostics GmbH, Mannheim, Germany) (24 (link)).

Primary antibodies against p53, Phospho-Akt (S473), Total-Akt, Phospho-GSK-3β, GSK-3β, Bax, Bcl-2, Bcl-XL, Mcl-1, Cox IV, Cleaved-Caspase3 and β-actin were purchased from Cell Signaling Technology. Lipofectamine™ Reagent was purchased from Invitrogen. HRP-conjugated anti-rabbit and or anti-mouse secondary antibodies an ECL-plus kit were from GE Healthcare. SC66 was purchased from Selleck Chemicals (Houston, TX). The oligonucleotide for shGSK-3 was synthesized as 5′-CCGGGTGTGGATCAGTTGGTAGAAACTCGAGTTTCTACCAACTGATCCACACTTTTT-3′. The CCK-8 kit was from 7 sea biotech (Shanghai, China).

The IC50 of TR on HaCaT and cell viability were determined by CCK8 kit (Lot. C008, 7Sea biotech, China) according to the manufacturer’s instructions. HaCaT cells were seeded into 96-well plates at the density of 5000 each well, and then treated with TR (Lot. S1439, Selleck) for 24 h with different concentrations. Next, cells were incubated with 100 (μL fresh medium with 10 (μL CCK8 solution for 2 h at 37°C and optical density (OD) was measured at 450 nm by Model 680 Microplate Reader (Bio-Rad, United States).

Dimethyl sulfoxide (DMSO) and 5-Aza-2′-deoxycytidine (5-Aza) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). DMEM and fetal bovine serum were purchased from Gibco BRL Co. (Grand Island, New York). Lipofectamine 2000 was purchased from Invitrogen Co. (Carlsbad, California, USA). The antibody for β-actin was purchased from Abgent Inc. (San Diego, California, USA). Antibodies for SYP and TET3 were purchased from Abcam (Cambridge, England). Maxima SYBR Green/ROX qPCR Master Mix was purchased from Genecopoeia and all of the primers used in this research were purchased from Genecopoeia. The related secondary antibodies and the ECL detection kit were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA). SYP and TET3 siRNA oligos and control siRNA oligos were purchased from Ribobio (Guangzhou, China). miR-27a-5p mimics, inhibitors and their control oligos were purchased from Ribobio. DAPI was purchased from Solarbia (Beijing, China). The EdU kit was purchased from Ribobio. The CCK8 kit was purchased from 7 sea biotech (Shanghai, China).

The CCK‐8 kit was bought from 7sea biotech and applied to evaluate the effects of PDCD10 on cell proliferation of U2OS and MG63 cells by the absorbance respectively. U2OS and MG63 cells were digested and seeded at 5000 cells per well respectively into 96‐well plates with five replicates. After culturing for 0, 24, 48, and 72 h, 10 μl CCK‐8 reagent was mixed with 100 μl fresh medium and added to each well. Then the plates were incubated for 2 h at 37°C to detect the absorbance of 450 nm. At last, the data were obtained for analysis.

Primary antibodies against p53, Phospho-Akt (S473), Total-Akt, PUMA, Phospho-FoxO3a (S253), Total-FoxO3a, Phospho-p65 (S536), Total-p65, Bax, Noxa, Bid, Bim, Bcl-2, Bcl-XL, Mcl-1, Cox IV, Cleaved-Caspase3, β-actin LC3, Total-MLKL, and Phospho-MLKL (Ser358) were purchased from Cell Signaling Technology. Lipofectamine™ Reagent was purchased from Invitrogen. HRP-conjugated anti-rabbit and/or anti-mouse secondary antibodies and ECL-plus kit were from GE Healthcare. Ipatasertib and 5-FU were purchased from APP Pharmaceuticals. afuresertib and perifosine were purchased from Selleck Chemicals(Houston, TX). Regofenib and cisplatin were purchased from Axon Medchem. Other chemicals were mainly from Sigma. CCK-8 kit was from 7 Sea Biotech (Shanghai, China). The plasmid of expressing PUMA was kindly supplied by Jian Yu, PhD^{9 (link)}.

Cell viability was evaluated by using a CCK8 kit (C008, 7Sea biotech, China) according to manufacturer's manual. Generally, PIG1 cells were seeded into 96-well plates at the density of 2 × 10⁴ cells per well before further treatments as indicated. Next, 10 μl of CCK8 solution was added to each well, and the cells were further incubated at 37°C for 2 h. The optical density (OD) was measured at 450 nm by Model 680 Microplate Reader (BioRad, USA). All experiments were performed in triplicate.