Control sirna a sc 37007

Rabbit anti-PHAX antibody (catalogue number ab171321), mouse anti-phosphorylated histone H3^S10 (pH3^S10) (catalogue number ab14955), and rabbit anti-phosphorylated histone H3^S10 (pH3^S10) (catalogue number ab5176) were all from Abcam Biotechnology, Cambridge, UK. Mouse anti-CD31 (PECAM1, 89C2; catalogue number 3528) and Hoechst-33342 were from Thermo Fisher Scientific, Renfrew, UK. Mouse anti-Cytokeratin (catalogue number VP-c420), TUNEL-label (dUTP^−FITC) (catalogue number 11767291910), and terminal transferase enzyme (TdT) (catalogue number 03333566001) were from Roche Diagnostics Ltd., Burgess Hill, UK. Anti-mouse-Cytokeratin (catalogue number sc-15367), human PHAX siRNA (a pool of 3 targeted-specific 19-25nts siRNAs (cat: sc-106785), individual siRNA duplex components (cat: sc-106785A; sc-106785B and sc-106785C), control siRNA (Fluorescein Conjugate)-A (sc-36869), and control siRNA-A (sc-37007) were all from Santa Cruz Biotechnology, Heidelberg, Germany. Sunitinib malate (PZ0012) was from Sigma-Aldrich Company Ltd., Gillingham, UK, dissolved in dimethyl sulfoxide (DMSO), and stored as aliquots at −20 °C until use.

Plasmids encoding FLAG-SFXNs were obtained from GenScript. Empty vector (pcDNA3.1D V5-His) was obtained from Invitrogen. HEK293T cells were seeded into 60 mm Petri dishes (1 × 10⁶ cells/dish) and transfected with 3 μg of DNA using Lipofectamine LTX (Life Technologies, Carlsbad, CA, USA) following manufacturer instructions. For RNA interference (RNAi) experiments, MCF7 cells were transiently transfected with either a scrambled control siRNA (Control siRNA-A sc-37007, Santa Cruz Biotechnology, Inc., Heidelberg, Germany) or a pool of specific siRNA for SFXN1 (sc-91814, Santa Cruz Biotechnology, Inc., Heidelberg, Germany). Transfection was performed using Interferin™ transfection reagent following manufacturer instructions (Polyplus-Transfection Inc., New York, NY, USA). Briefly, a mix of siRNA and Interferin™ transfection reagent was incubated for 10 min at room temperature (RT) and added to each well at a final concentration of 10 nM. Cells were incubated at 37 °C under standard culture conditions and collected after 3, 4, or 7 days to extract proteins.

In all transfection experiment, cells were initially seeded at 50–60% confluency in a six-well plate and incubated for 8 h in growth media. The cells were transfected using the jetPRIME transfection reagent (Polyplus transfection, France) according to the manufacturer’s protocols. Small interfering RNAs (si-RNA) PDHA1 (sc-91064 and 5160), si-PKM2 (sc62820 and 5315), and control si-RNA-A (sc-37007) were purchased from Santa Cruz and Bioneer. Si-RNA GPR174 (84636-2), si-RNA Il13Ra2 (3598-1), si-RNA KDM1B (221656), and si-IR-A (customized sequence; sense strand 5′ CUAGUCCUGC-AGAGGAUUU- 3′ and antisense strand 5-′ AAAUCCUCUGCAGGACUAG- 3′) [37 (link)] were also bought from Bioneer (Daejeon, Republic of Korea). These si-RNAs were transfected at a final concentration of 30–100 nM, and 2–3 µg DNA was transfected to the cells. Briefly, si-RNA and DNA were diluted into in 200 µL of the jetPRIME buffer, then 2–9 µL of the jetPRIME reagent was added, and the mixture was incubated for 10 min. Finally, the solutions were added to the six-well plate and incubated for another 24–48 h before performing the stated experiments.