Dharmafect 1 reagent

To silence the expression of genes of interest, a set of ON-TARGET human siRNAs against CTNNA1 (Horizon Discovery, J-010505-06), CDH5 (Horizon Discovery, J-003641-07) or untargeting control were used (Horizon Discovery, D-001810-01), using previously defined conditions^{53 (link),54 (link)}. Briefly, HUVECs were seeded the day before the transfection at a concentration of 1.8 × 10⁵ cells/mL to reach 60–70% confluence on the day of the transfection. Then, cells were transfected with 25 nM of siRNA using Dharmafect 1 reagent (Horizon Discovery) following the Dharmacon siRNA transfection protocol. The cell culture medium was replaced 24 h after transfection by fresh complete medium and cells were kept under culture conditions up until 72 h post-transfection and then processed for further experiments.

ON-TARGETplus SMARTpool-Human of four specific POLA1 (#L-020856-00-0005) and POLE (#L-020132-00-0005) siRNAs (Horizon Discovery Lafayette, CO, USA) were used to transiently transfect cells at 70% of confluence with Dharmafect 1 reagent (Horizon Discovery) as per manufacturer’s protocol. Non-targeting control siRNA (#D-001810-10-20) were used as negative controls. After 24 h, cells were trypsinized and plated at the required density into 96-well plates (for XTT assays) or 6-well plates (for immunoblot assays). The next day cells were treated with appropriate inhibitors and incubated for 48 h for XTT assays or overnight prior to cell harvest and protein lysate preparation for immunoblots. The sequences of siRNAs used in this study are listed in Supplementary Data 8.

Rat cardiac myoblast (H9c2, 2-1) cells were acquired from the American Type Culture Collection (Manassas, VA) and cultivated in Dulbecco’s Modified Eagle Medium (GIBCO, Dublin, Ireland) containing 10% fetal bovine serum (HyClone; GE Healthcare, Bucks, UK), 100 U/mL penicillin, and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA), in a humidified atmosphere with 5% CO₂ at 37°C. The cells were cultured until 70–80% confluence and were then serum deprived for 24 h before each experiment. The cells were incubated with 0, 0.25, or 1.0 mM IS or 0, 50, or 100 ng/mL recombinant mouse FGF23 (2629-FG; R&D Systems, Minneapolis, MN) diluted in normal saline (NS) and collected after 24 and 72 h for real-time reverse transcription-polymerase chain reaction and western blotting, respectively. To perform small interfering RNA (siRNA) knockdown, the cells were transfected with On-TARGETplus SMARTpool siRNA [non-targeting control, aryl hydrocarbon receptor (AhR), and FGF23; Horizon Discovery, Cambridge, UK] using Dharmafect 1 reagent (Horizon Discovery) in accordance with the manufacturer’s instructions.

SiRNA duplexes were synthesized by Dharmacon with 3’UU overhangs, except for siRNA pool targeting Psd3/EFA6D which was purchased from Santa Cruz Biotechnology (sc-77475). Non-targeting siRNA control was from Ambion (siControl 1). siRNAs were transfected into HeLa cells grown on 96 well plates using Dharmafect-1 reagent (Dharmacon) according to the manufacturer’s recommendations. The replication experiments were performed ~72h post siRNA transfection except for cells treated with siRNA targeting GBF1 which were first assessed at 48h post-transfection because prolonged depletion of GBF1 induces significant cytotoxicity. Cell viability was determined with CellTiter-Glo assay (Promega).

B16-F10.9-4 cells (7 × 10⁴ cells/ml) or JC TetON LIP cells (3 × 10⁴ cells/ml) were cultured for 24 h in media lacking penicillin and streptomycin, transfected for 24 h with siRNA pools (Dharmacon, Lafayette, CO, USA) directed against murine c-Jun (L-043776-00-0005), c-Fos (L-041157-00-0005), ATF2 (L-042961-01-0005), ASK1 (M-041179-01-0005), Ogn (L-058799-01-0005), C/EBPβ (L-043110-00-0005), CHOP (L-062068-00-0005) or p53 (L-040642-00-0005) mRNAs, using DharmaFECT 1 reagent (Dharmacon) according to the manufacturer’s protocol. Then, expression of LIP was induced by Doxy for 16-24 h. ER stress was induced by treating cells for 18–24 h with Tm, BFA or Tg. Cells treated with ON-TARGETplus non-targeting siRNA pool (D-001810-10-20) or siGenome non-targeting siRNA pool (D-001206-14-05), were used as negative control.

Parental and osteotropic MDA-231 cells were transfected with siRNA (25 nM) using Dharmafect 1 reagent (Dharmacon, CO, USA), according to the manufacturer’s instructions. Small interfering RNAs (siRNA; siGenome SMART pool) as a pool of four annealed double-stranded RNA oligonucleotides for IKKβ (M-003503-03), IKKε (M-003723-02), TRAF2 (M-005198-00-0005), and non-targeting control no. 3 (D-001210-03-05) were used according to the manufacturer’s instructions. For transfections involving the combination of TRAF2 and IKKβ oligonucleotide, each oligonucleotide was transfected at a concentration of 50 nM, so the total siRNA concentration is 100 nM. The cells were cultured for 48 hours in antibiotic free complete medium with the transfection reagent. The efficiency of TRAF2, IKKε and IKKβ knockdown and overexpression was assessed by Western blot analysis.