Non specific control sirna

Antibodies against ERK5, phospho-ERK5, ERK1/2, phospho-ERK1/2, FAK, N-WASP, Gelsolin, smad3, and p-smad3 were purchased from Cell Signaling Technologies. The antibodies to detect p-PLK1, USF1 were obtained from Abcam (Cambridge, MA, USA). The antibodies against SPA1, E-cadherin, Snail, N-cadherin, Fibronectin, Vimentin, and α-tubulin were from Santa Cruz Biotechnology. Anti-p-USF1 was from ThermoFisher Scientific. The antibodies to detect pSer⁹¹⁰ and pTyr³⁹⁷of FAK were from BioSource (Camarillo, CA, USA). Human ERK5 siRNA, USF1 siRNA, and non-specific control siRNA was commercially obtained from Santa Cruz Biotechnology (USA).

30 pmol of mouse Smad2/3 silencing RNA (siRNA) (Santa Cruz), or p38α siRNA (Cell Signaling), p38β siRNA (Cell Signaling), or non-specific control siRNA (Santa Cruz, Cell Signaling) were transfected into ATDC5 cells using Lipofectamine RNAi (Thermo Fisher Scientific) as per manufacturer’s protocol. Then after 48 hours of transfection, cells were treated with TGF-β1 or vehicle control. FITC-conjugated control siRNA (Santa Cruz) was used to test for transfection efficiency, approximately 80–90% of cells were transfected with siRNA.

USP17 siRNA and nonspecific control siRNA were purchased from Santa Cruz Inc. MDA-MB-231 cells were plated at 2.5 × 10⁵ cells/well in 6 well plates. Double-stranded siRNAs were transfected into MDA-MB-231 cells using the INTERFERin transfection kit (Polyplus transfection Inc., Illkirch, France) according to the manufacturer's instructions. 24 hours after transfection, cells were subjected to Western blot analysis or treatment and WST-1 cell viability assay.

We used an established mouse model that utilized warm hepatic ischemia followed by reperfusion.^{2 (link)} Mice were injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the arterial/portal venous blood supply to the cephalad liver lobes. After 90 min of ischemia, the clip was removed, and mice were sacrificed after 6 h of reperfusion. Some animals were injected via the tail vein with bone marrow-derived macrophages (BMMs, 5 × 10⁶ cells in PBS/mouse) transfected with lentivirus-expressing HSF1 (Lv-HSF1) 24 h prior to ischemia, and some animals were injected i.v. with Snail siRNA or nonspecific (control) siRNA (2 mg/kg) (Santa Cruz Biotechnology, CA) mixed with mannose-conjugated polymers (Polyplus transfection™, Illkirch, France) at a ratio defined according to the manufacturer’s instructions 4 h prior to ischemia, as described.^{2 (link)} Some animals were injected i.v. with recombinant JAG1 (0.5 mg/kg, R&D Systems) or PBS 1 h prior to ischemia.

Predesigned double-stranded small interfering RNAs (siRNAs) from Integrated DNA Technologies were used; 30 pmol of human Smad3 silencing RNA (siRNA) (Santa Cruz Biotechnology, TX, USA) or nonspecific control siRNA (Santa Cruz Biotechnology) were transfected into hBMSCs using Lipofectamine RNAi (Thermo Fisher Scientific) according to the manufacturer’s protocol. Then, after 48 hours of transfection, cells were treated with TGFβ3 or vehicle control. FITC-conjugated control siRNA (Santa Cruz) was used to test for transfection efficiency, and approximately 80-90% of cells were transfected with siRNA.