Dharmafect 2 reagent

Cells were seeded in 24-well plates 48 hours before siRNA transfection in order to have 60–70% confluency on the day of transfection. Cells were transfected with siRNA (Dharmacon) targeting (ATF4 and DDIT3) and control siRNA at a final concentration of 100 nM using DharmaFECT 2 reagent (Dharmacon). ON-TARGET plus siRNA pool (Dharmacon) was used for all knockdown experiments because it contains a pool of four individual siRNA sequences for the target gene. RNA knockdown was confirmed by assessing mRNA levels with qRT-PCR.

STAT1 was silenced in MDA-231 cells by using small interfering RNA (siRNA) at 5 µM (STAT1-RNAi sense 5′-(P)CUACGAACAUGACCCUAUCUU-3′, anti-sense 5′-(P)GAUAGGGU CAUGUUCGUAGUU-3′, Dharmacon, Thermofisher Scientific) transfected with Dharmafect2 reagent according to the manufacturer' instructions. siGenome non-targeting RNApool2 (Dharmacon) served as controls at the same concentration. Proteins were extracted after 48 h and analyzed by immunoblotting.

BT549, MDA‐MB‐468 and MDA‐MB‐436 cell lines, which all belong to triple‐negative breast carcinoma cells, were purchased from Cell Bank of the Institute of Basic medicine, Chinese Academy of Medical Sciences (Beijing, China), or obtained as NCI‐ICBP45 kit procured through American Type Culture Collection (ATCC; ATCC Breast Cancer Cell Panel, Manassas, VA, USA). BT549 cells were cultured in RPMI 1640 medium (Boster, Wuhan, China) supplemented with 10% FBS (Gibco, Carlsbad, USA), 1% antibiotics and 0.023 IU·mL⁻¹ bovine insulin (Sigma‐Aldrich, Saint Louis, MO, USA). MDA‐MB‐436 and MDA‐MB‐468 cells were cultured in Dulbecco's modified Eagle's medium (Boster) with 20% or 10% FBS (Gibco) and 1% antibiotics. All cells were kept at 37 °C and 5% CO₂. The short hairpin RNA (shRNA) product of SGLT1 (sh‐SGLT1) with a targeted sequence of ATCTTTCTCTTATTGGCAA and its negative control (sh‐NC) were purchased from GeneChem (Shanghai, China) and transfected into cells according to manufacturer’s instructions. Short interfering RNA (siRNA) oligos against SGLT1 (MU‐007589‐01‐0002) were purchased from Dharmacon (Lafayette, CO, USA). Sequences are available from Dharmacon or upon request. As a negative control, we used siGENOME RISC‐Free siRNA (Dharmacon). Cells were transfected with the indicated siRNA oligos at a final concentration of 35 nm using Dharmafect 2 reagent (Dharmacon).

Validated pools of FBXW7α-, MCL1-, PLK1-siRNA and non-targeting siRNA as negative control were obtained from GE Dharmacon (ON-TARGETplus SMART pools L-004264, L-004501, L-003290, and D-001810). Transfections were carried out using DharmaFECT 2 reagent (GE Dharmacon) according to manufacturer's instructions. All siRNA pools were used at 50 nM. Cells were subjected to different treatments 24 h after silencing. Transient transfections of pCMVHA and pCMVHA-FBXW7 plasmids [28 (link)] were carried out using FuGENE reagent (Promega) according to manufacturer's instructions. Cells were subjected to different treatments 24 h after transfection.

Small interfering RNAs (siRNA) targeting siRNA control (ON TARGETplus control siRNA, 100 nM) and VEGFR2 (ONTARGET plus smartpool, 100 nM) (Dharmacon, Inc., UK) smartpools were transfected into H441 cells using DharmaFECT 2 reagent (4 μl/ml, Dharmacon, Inc.) according to manufacturer's instructions. For the proliferation assay end point, cells were incubated in the transfection media for 24 h. The transfection media was removed and replaced with 100 μl of fresh 0.2% FBS DMEM/F12. DMEM/F12 treated with ± FBS or VEGF was added and plates were incubated for a further 4 days. At this point the cells were stained with crystal violet, allowed to dry and eluted with glacial acetic acid. The absorbance was read at 590 nm using the POLARstar Omega plate reader.