Am16708

The SMARTpool siRNA targeting YAP (YAP siRNA‐1) and a non‐targeting siRNA used as control were purchased from Thermo Scientific Dharmacon (Pittsburgh, PA, USA). Another YAP siRNA (AM16708) targeting the 3′UTR end of the YAP gene (YAP siRNA‐2) was purchased from Life Technologies (Grand Island, NY, USA). The YAP plasmid DNA (pcDNA4/HisMaxB‐YAP) used to overexpress the YAP gene in the cells was purchased from Addgene (Cambridge, MA, USA).
The mesothelioma cells were plated in 6‐well plates (for Western blot or PCR) or 24‐well plates (for reporter assay) 24 hours before transfection. Cells were transfected with 100 nmol/L of siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA). For the YAP rescue procedure with YAP forced overexpression, cells cultured in 6‐well plates were transfected with 4 μg/well of YAP plasmid DNA, and those in 24‐well plates were transfected with 0.8 μg/well. After transfection for 48 hours, cells were harvested for further analysis.

Human aortic SMCs (PromoCell, C-12533, 3 different donors) were cultured on 6-well plates (1.5×10⁵ cells/well) using SMC Growth Medium 2 (PromoCell C-22062) until 70% confluent. MFAP4 siRNA (50 nmol/L, Life Technologies, AM16708, Assay ID: 11386) and control siRNA (50 nmol/L, Life Technologies, AM4613) were transfected by using lipofectamine in different passages of different donors (2 for donor 1, 7 for donor 2, and 5 for donor 3) for 6 hours and subsequently in M199 with 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5% fetal bovine serum overnight. After replaced with complete medium (M199 with 100 U/mL penicillin, 100 µg/mL streptomycin, and 20% fetal bovine serum) and cultured for 48 hours, the cells were washed using M199 3 times, and further cultured in M199 for 24 hours. The cells were harvested for RNA extraction as detailed below. In total, 14 individual experiments using 3 different cell lines were performed for each condition. The experiment was repeated with a different MFAP4 siRNA at a lower concentration (25 nmol/L, Life Technologies, 4392420, Assay ID: s8716).

U-87 GBM cells at ∼70% confluency were seeded on a 6-well plate and transfected after 24 h with 100 nM siRNA against PPAR-γ (#AM16708, siRNA ID 143093, Invitrogen by Thermo Fischer Scientific, Waltham, MA USA) or 100 nM control siRNA (#AM4611, Invitrogen by Thermo Fischer Scientific, Waltham, MA USA) for 6 h using Lipofectamine transfection reagent (#18324-020, Invitrogen by Thermo Fischer Scientific, Waltham, MA USA) as previously described [5 (link)]. Briefly, two mixes were prepared: a mix of normal culture medium (DMEM—Sigma-Aldrich^® Catalog No. D5030; St. Louis, MO, USA) with 100 nM control siRNA and a mix of normal culture medium (DMEM—Sigma-Aldrich^® Catalog No. D5030; St. Louis, MO, USA) with 100 nM PPAR-γ siRNA; both mixtures were incubated for 5 min at room temperature. Next, the prepared solutions were mixed with the Lipofectamine transfection reagent and incubated for 15 min according to the manufacturer’s instructions. The mixture was added to each well (250 μL/well) that already containing 1 mL of DMEM. After 6 h of incubation, the cells were treated for 24 h with SP at a concentration of 50 or 100 mM while the normal culture medium was added to the siRNA control group and PPAR-γ siRNA (Scheme 2).

Pre-designed siRNA constructs were used for RNA interference, procured from Invitrogen (AM16708; Assay ID 102888) along with a scrambled siRNA (4390843). Transient silencing of UBA52 was performed using lipofectamine 3000, as described in the transfection kit (L3000-001).

We used Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) to transfect VICs with siRNA. We followed the protocol that was provided by the manufacturer. The siRNA for HIF-1α (AM16708, ID: 106498) and HIF-2α (AM16708, ID: 106446) and silencer negative control #1 (4390843) were purchased from Invitrogen. To confirm the efficiency of silencing we performed Western blot analysis. Experiments were repeated at least three times.