Sirna transfection reagent

Knockdown of PHD1 and VHL were performed using specific siRNAs purchased from GenePharma Company (Shanghai, China). Sequences of siRNAs are as follows: siPHD1-A, 5′-GCAUCACCUGUAUCUAUUATT-3′; siPHD1-B, 5′-CCAACAUCGAGCCACUCUUTT-3′; siPHD1-C, 5′-GCUAGCAUCAGGACAGAAATT-3′; siVHL-A,5′-CCACCCAAAUGUGCAGAAATT-3′; siVHL-B, 5′-GUCUCAUUCUCAGAGUAAATT-3′; siVHL-C, 5′-AACUGAAUUAUUUGUGCCAUCTT-3′. A scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) was used in parallel experiments as negative control. siRNAs were transfected to SKOV3 and 3AO cells at 40–50% confluence with siRNA transfection reagent (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Effective siRNAs were screened out according to the results of real-time RT-PCR after 48 h incubation and by western blot after 72 h, respectively. Then the effective siRNAs were transfected into cells and incubated for 24 h followed by hypoxia induction with or without 20(S)-Rg3 treatment for another 24 h. The effect of PHD1 and VHL silence on suppression of HIF-1α by 20(S)-Rg3 was assessed at protein level.

6T-CEM cells were cultured in 24-well plates with a 5~7^∗10⁵/ml density in RPMI1640 medium containing 10% FBS. The cells were then transfected with siRNA targeting lncRNA ENST00000502883.1 (sequence: 5′-CAAACGUUCAUGUGAAAGATT-3′; 5′-UCUUUCACAUGAACGUUUGTT-3′) and synthesized by GenePharma, Shanghai, China, using the siRNA transfection reagent (Roche, Penzberg, Germany) for 48 h. The scrambled siRNA (GenePharma) was used as a negative control. Six hours before the end of the experiment, 80 nM of PMA, 1 μg/mL of ionomycin and10 μg/mL of Brefeldin A was added to the culture. The efficiency of knockdown was examined by qRT-PCR. The expression of target gene CXCL16 was detected by qRT-PCR and Western blot.

Following the manufacturer’s instructions, pcDNA3.1(+)-STAT3WT, pcDNA3.1 (+)-STAT3Y705F, pcDNA3.1-Beclin1 were transiently transfected into cells with Lipofectamine^TM 2000 (Invitrogen) for 48 h before incubation in culture medium.
Atg14 siRNA (CST), Beclin 1 siRNA (CST) or control siRNA (CST) were transfected into cells using siRNA transfection reagent (Roche) according to the manufacturer’s instructions. Then, cells were incubated for 72 h before treatments were administered.