Ow cytometer

The total RNA was extracted from the cancer cells using the RNeasy plus mini kits (Qiagen, Germany). Reverse transcription was performed using the PrimeScript RT Master Mix (Takara, Japan). qRT-PCR was performed using the SYBR green mix (Toyobo, Japan) with the CFX96TM Real-time PCR Detection System (Bio-Rad, USA). The 2 -ΔΔCt method was used to calculate the relative expression. 36B4 was used for internal control. All assays were performed in triplicates.
Proliferation, cell cycle and colony formation assay CAL62 and KHM-5M were treated with SiTRIM11 or SiControl in 6-well plates. Forty-eight hours after transfection, the cells were counted and 2000 cells were seeded in 96-well plates. Cell viability was measured using Cell Counting Kit-8 (CCK8) every 24 hours. Cells transfected with SiTRIM11 or SiControl for 48 hours were stained with propidium iodide (Multisciences, China) and analyzed by ow cytometer (Beckman, USA), The cell cycle phases were determined by relative DNA content. For the clone formation assay, cells were treated with 50 nM TRIM11 siRNA or 50 nM siControl, after 48 hours, the cells were typsinized and seeded (1-1.5×103 cells/well) in 6-well plates and maintained in complete medium for 2 weeks. The cells were xed with 4% paraformaldehyde for 2 hours, and stained with 1% crystal violet. EdU incorporation assay was performed as our previously described.

Apoptosis of cells was determined by an Annexin V-FITC Apoptosis Detection Kit (Nacalai, Tesque, Japan). The treated cells were cultivated at 37℃ with 5% CO 2 for 48 hours, centrifuged at 1000 rpm for 3 minutes, washed in PBS for twice, and then centrifuged again. Then, 20 μL Annexin V Binding Buffer (10 ×) was added into 200 μL PBS. Cells were resuspended in PBS, and then lled with 10 μL Annexin V-FITC conjugate and 5 μL PI solution, and then cultivated at room temperature in the dark for 15 minutes. Then the cells were added with 300 μL binding buffer, and the excitation wavelength at 488 nm was determined using a ow cytometer (Beckman Coulter, USA).

The cells (10 6 /tube) were washed twice with phosphate-buffered saline (PBS) and incubated with PEanti-human CD44 and FITC-anti-human EpCAM (appropriate dilution per antibody) at 4°C for 20 min.
Subsequently, the cells were washed with PBS twice. The labeled cells were analyzed using a ow cytometer (Beckman Coulter, Inc., Brea, CA, USA).

Autophagy in live cells was analyzed using the Cyto-ID autophagy detection kit (Enzo, NY, USA) with proprietary probes speci cally staining autophagosomes according to the manufacturer's protocol. Brie y, the cells were harvested, resuspended in 500 μL of freshly diluted Cyto-ID green detection reagent and incubated at room temperature for 30 min in dark. Then uorescence intensity was analyzed using a ow cytometer (Beckman, CA, USA).

Cell apoptosis was detected using the following methods: (1) The cells were xed with 4% paraformaldehyde for 10 min and stained with Hoechst 33258 (Beyotime) for 10 min in dark. Then the cells were photographed under a uorescence microscope (OlympusIX81) and the apoptotic cells were characterized by the nuclear morphology changes. (2) The cells were harvested and stained with Annexin-/FITC and PI (BD, California, USA) at room temperature for 15 min in dark. The apoptotic cells were analyzed under a ow cytometer (Beckman).

The cells were seeded in a 6-well plate at a density of 2 10 5 cells/well, with the complete medium overnight. The cells were collected, washed by ice-cold PBS for three times and then resuspended by 1 × binding buffer. 100 µL cell suspension was stained by 1/1000 (v/v) Annexin V-FITC for 20 min at room temperature, in the dark. Next, the cells were washed thoroughly by PBS and stained with 2 µg/mL propidine iodide (PI) in deliquated binding buffer. The apoptosis evaluation was performed by ow cytometer (Beckman Coulter, CA, USA) and analyzed by FlowJo software (RRID:SCR_008520) (Tree Star, OR, USA).
To assess the cell proliferation, the EdU (5-ethynyl-2'-deoxyuridine) labeling assay was performed by incubating NC and HepG2/NFAT2 cells with serum free medium containing 10 µmol/L EdU for 2 h. Then, the cells were washed by PBS and xed for 15 min at room temperature. The xed cells were washed and re-suspended in permeabilization buffer for 10 min in dark place. The cell suspension was incubated in Click-iT plus reaction cocktail for 30 min at room temperature in the dark. The stained cells were washed and analyzed by ow cytometer.

The cells (10 6 /tube) were washed twice with phosphate-buffered saline (PBS) and incubated with PEanti-human CD44 and FITC-anti-human EpCAM (appropriate dilution per antibody) at 4°C for 20 min.
Subsequently, the cells were washed with PBS twice. The labeled cells were analyzed using a ow cytometer (Beckman Coulter, Inc., Brea, CA, USA).