Ow cytometry

The Kyse450 cells transfected with MCTP1 siRNA or control siRNA were seeded into 6-well plates, harvested after 48h and rinsed with PBS twice. Cells were treated with 200μl binding buffer, 5μl Annexin V-FITC and 5μl propidium iodide (PI). After incubation in the dark for 30min at room temperature, the cell apoptotic rate was measured by ow cytometry (Beckman) and analyzed by Flowjo Software. The experiments were performed independently three times, and a representative is shown.

Lentiviral vector plasmids and packaging plasmids (psPAX and pMD.2G) were purchased from Addgene.
Lentiviral particles carrying pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1 and pCDH-EF1-GLP1 were produced through the transfection of HEK293T packaging cells with a 3rd generation plasmid system. HEK293T cells were transfected with 24 µg of plasmids, 48 µl of Lipofectamine LTX and 24 µl of PLUS reagents, and the proportions of the pMD.2G, psPAX, and pCDH-EF1 plasmids were 1:2:3. The supernatants were collected at 24 and 48 h after transfection, ltered through 0.45-μm lters, and harvested by ultra ltration with a 100-kDa spin column (Millipore) at 4 °C and 4,000 g for 30 min. The lentiviral particles were aliquoted and stored at -80 °C until use. The transfection e ciency was determined based on EGFP expression using ow cytometry (Beckman), and the viral titers were determined according to the following equation: virus titer (pfu/mL) = cell number in each well × virus dilution factor × 10/ volume of added virus uid (mL).

A 6-well cell culture plate was inoculated with 1×10 6 RAW264.7 cells per well and incubated for 16 h. WT, ΔsseK3 mutant and sseK3-complemented strains were coincubated with RAW264.7 cells at a multiplicity of infection (MOI) of 100:1, with three replicate wells per strain. To allow the bacteria to fully contact the RAW264.7 cells, the plates were centrifuged with 1000 rpm/min. Then, gentamicin-containing medium (100 μg/mL) was added and incubated at 37 ℃ with 5% CO 2 . After incubation, the supernatants were aspirated, and the cells were washed three times with PBS. The percent of cells undergoing apoptosis was detected by ow cytometry using Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH Jiangsu China). The cells of infected and mock groups were digested with 0.25% trypsin and washed three times with ice-cold phosphate buffered saline (PBS) and suspended in Binding Buffer with 500 μL, followed by adding 5 μL Annexin V-FITC and 5 μL Propidium Iodide (PI). Then the solution was placed in the dark room for 15 min at room temperature followed by immediately analysis using ow cytometry (Beckman Coulter, Inc., Fullerton, CA, US).

Cell apoptosis was measured using PE Annexin V Kit I (BD Biosciences, NJ, USA). Briefly, cells were collected and resuspended in 1× binding buffer. Thereafter, the solution (1×10 5 cells) supplemented with 5 μL of PE Annexin V and 7-AAD was incubated in the dark for 15 min at room temperature. The apoptotic cells were identied by ow cytometry (Beckman Coulter, CA, USA). All the experiments were performed in triplicate.

Following conventional digestion, cells in the logarithmic growth phase were used to prepare a single cell suspension; 2 × 10 5 cells/mL were seeded in 6-well plates. After treatment for 48 h, all cells were collected and centrifuged at 2000 rpm for 5 min; the supernatant was discarded after washing with PBS, and centrifugation was repeated twice. The cells were stained with uorescein FITC-conjugated Annexin V and PI (40302ES60, Yeasen, Shanghai, China) and analyzed by ow cytometry (Beckman Coulter, Brea, CA, USA). The ratio of both early and late apoptosis cells was calculated [22] .

The cells were cultured for 24 h, then collected and resuspended once with pre-cooled 1×PBS (4°C). The cells were centrifuged at 1000 rpm for 5-10 minutes. Then 300 μl of 1× Binding Buffer was used to suspend the cells. 5μl of Annexin V-FITC was added into cells. And the cells were incubated for 15 minutes at room temperature. 5 minutes before the detection, the 5μl of PI staining was added and then 200μl of 1×Binding Buffer was added. Samples were nally detected by ow cytometry (Beckman Coulter, Brea, CA, USA) and analyzed by Cell Quest software (BD Bioscience, San Diego, CA).