The results in Figure 2B showed that knockdown of ANRIL could significantly increase the rate of apoptotic cells in both MKN-45 and SGC-7901 cells compared to the shNC group (both P< 0.001).
After cell transfection, the expression of miR-99a in cells transfected with shANRIL was significantly higher than that of the shNC group in MKN-45 and SGC-7901 cells (both P<0.01, Figure 3).
Further experiments showed that combination of ANRIL silence and miR-99a overexpression down-regulated BMI1 expression, whereas BMI1 level in cells transfected with shANRIL and miR-99a inhibitor was nearly the same as that of the shNC + mimic control groups (Figure 5D).
shANRIL: pENTR[TM]/U6 vector carrying small hairpin RNA targeting ANRIL; shNC: pENTR[TM]/U6 vector carrying a non- targeting sequence; qPCR, quantitative real-time PCR; CCK-8, Cell Counting Kit- 8.
6 x [10.sup.5] HTR8/SVneo cells (IL-33 shNC group or scrambled shRNA group) were used for the invasion assay, and 2 x [10.sup.5] HTR8/SVneo cells (IL-33 shNC group or scrambled shRNA group) were used for the migration assay; both were suspended in 500 [micro]l of medium with 1% FBS and then separately placed in the upper chamber, whereas 750 [micro]l of medium with 10% FBS was added to the lower chamber.
8 x [10.sup.3] HTR8/SVneo cells (blank group, IL-33 shNC group, or shIL-33 group) in 100 [micro]l of medium were put into 96-well plates and incubated for 12 hr to allow the cells to adhere before the assay (five wells for each group and three groups on each plate).
1 x [10.sup.5] of HTR8/SVneo cells (normal group, IL-33 shNC group, or scrambled shRNA group) were seeded into six-well plates.
Supplemental Figure 1: immunofluorescence images demonstrated the expression of ST2/IL-1 R4 in the shNC group and the shIL-33 group.
The protein used for western blotting was extracted from MSCs with pBABE-puro, pBABE-PDK2, shNC and shPDK2 using RIPA lysis buffer (Beyotime Institute of Biotechnology, China).
The pBABE-puro and pBABE-PDK2 were transfected to construct PDK2 overexpression and shRNA-PDK2, shNC were transfected to construct PDK2 knockdown with lentivirus in MSCs, respectively.
We found that overexpressing PDK2 enhanced cell viability and caused a striking decrease on day 9 (P<0.05, Figure 2A), while cell viability was significantly reduced by shPDK2 compared to shNC at 9 days (P<0.05).
mRNA expressions of these markers were significantly lower with the addition of shPDK2 than with shNC (all P<0.05).