Control sirnas sc 37007

Control siRNAs (sc-37007 and SN-1002) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and Bioneer (Daejeon, Korea), respectively. For siRNA specific to MAGEA12, predesigned and validated MAGEA12 siRNA (sc-108017) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Customized MAGEA12 siRNA designed with Invivogen design tool (https://www.invivogen.com/sirnawizard/, accessed 21 May 2021) was synthesized by Bioneer (Daejeon, Korea). The sense and antisense sequences for customized MAGEA12 siRNAs are as follows: 5′-GCUUCCAAGUAGCACUCAGUA-3′ and 5′-UACUGAGUGCUACUUGGAAGC-3′. Cells were seeded and incubated until they reached 60–80% confluence (12–18 h). For transfections, lipofectamine RNAiMAX transfection reagent (Invitrogen, Waltham, MA, USA, 13778150) was diluted in Opti-MEM I reduced serum medium (Gibco, Waltham, MA, USA, 31985-070) and gently mixed with siRNA (10 μM) diluted to a final concentration of 30 nM in the same medium. After a 5 min incubation at room temperature (RT), the resulting siRNA-lipid complexes were added to the cells and incubated for 1–3 days at 37 °C.

MPH were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum and antibiotics at 37 °C in an atmosphere with 5% CO₂. PNPLA8 specific- siRNA (SR309408; Origene, USA) and control siRNAs (sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cells were transfected for 24 h with 10 nM of each siRNA and Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions; subsequently, plates and chamber slides were washed and refilled with fresh media with or without serum for 24 h followed by analysis.

We obtained validated siRNAs targeting human EP2 (sc-40171) or Gαs (sc-29328) mRNA, as well as control siRNAs (sc-37007) from Santa Cruz Biotechnology (TX, USA). The control siRNA had a sequence that did not target any known mammalian genes. We incubated human MSCs with 100 pMoles of siRNA against EP2 (siEP2) or Gαs (siGαs) and control siRNA (siCTRL) with Lipofectamine 3000 (Invitrogen, CA, USA) for 6 hr. Then, we changed the medium and further cultivated cells for 48 hr. We evaluated the down-regulation of target mRNA by semi-quantitative RT-PCR.

Kunming mice (SPF class) were purchased from the Center of Experimental Animal of the Zhengzhou University. The rhabdomyosarcoma (RD) cell line, derived from a human rhabdomyosarcoma, was purchased from ATCC and cultured in Dulbecco's modified Eagle medium (DMEM) (Corning, NY) with 4.5 g of glucose/L and 10% fetal calf serum. The cells were maintained in our lab. The echovirus type 9 strain was isolated from the cerebrospinal fluid of a meningitis patient. Quercetin and rabbit polyclonal antibodies (pAb) against GAPDH were purchased from Sigma Chemicals (St. Louis, MO) and Hangzhou GoodHere Biotechnology Co. Ltd (Hangzhou, China), respectively. Rabbit monoclonal [EPR16892] to Hsp70 antibody and Goat anti-Rabbit IgG H&L (HRP) were purchased from Abcam (Shanghai, China). HSP 70 siRNA (h) (sc-29352) and Control siRNAs (sc-37007) were purchased from Santa Cruz Biotechnology, Inc.