Pten sirna

PTEN clone 1 was purchased from GenScript, USA (Clone ID: M12583), and PTEN clone 2 was provided by Prof. CN Kundu, KITT School of Biotechnology, Bhubaneswar, India; vFLIP K13-GFP and PCDNA were gifted by Dr. Patricia S. Steeg, National Cancer Institute, and Dr. Matthew B Rettig, UCLA School of Medicine, United States. PTEN siRNA was purchased from Invitrogen (4427037 SIL SEL SiRNA INV STD, 1 NM; Assay ID_s534589; 29349990 1).

Rhabdomyosarcoma (RD) and human embryonic kidney (HEK) 293 cells were obtained from American Type Culture Collection (ATCC, USA). Unless specified otherwise, all cells were cultured at 37°C in a humidified 5% CO₂ incubator in minimum essential medium (MEM, Gibco) supplemented with 2 or 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco). RD cells or HEK293 cells were transiently transfected with miR-494 mimics (MC12409), miR-494 inhibitors (MH12409), miRNA negative control (miR-NC), mutant PTEN siRNA (CST, #6201), and PTEN siRNA (CST, #6538) using Lipofectamine 2000 (Invitrogen). The miRNAs and siRNAs were commercially purchased by Thermo Fisher and Cell Signaling, respectively.

Transfection of miRNAs to cell lines was achieved using Lipofectamine® 2000 (Invitrogen). Briefly, 20 μM miR-19b mimics or controls (GenePharma, Shanghai, China) were transfected to 293T cells, Jurkat cells, or Clone-X cells according to the protocol provided by the manufacturer. In primary cells, Lipofectamine® RNAiMAX (Invitrogen) was used for the transfection according to the protocol provided by the manufacturer. Briefly, 20 μM miR-19b mimics, inhibitors (GenePharma), or controls were transfected to isolated CD8⁺T or CD4⁺T cell-depleted PBMCs. In addition, isolated primary CD4⁺ T cells from healthy controls were transfected with 20 μM miR-19b mimics or controls. The forced reduction of phosphatase and tensin homolog (PTEN) was achieved by introducing 20 μM PTEN siRNA (Invitrogen) to isolated CD8⁺T cells. The siRNA control used in this experiment was non-specific Stealth RNAi® Negative Control Duplexes. The sequences of the mimics and inhibitors are listed in Supplemental Table 3.

Pten siRNAs and negative scrambled control siRNA were purchased from ShangHai GenePharma. We screened siRNA-Pten-a (sense 5′-GGG UAA ACA CAU UCU UCA UTT-3′; antisense 5′-AUG AAG AAU GUG UUU ACC CTT-3′), siRNA-Pten-b (sense 5′-CCA GAG GCU AGC AGU UCA ATT-3′; antisense 5′-UUG AAC UGC UAG CCU CUG GTT-3′), and siRNA-Pten-c (sense 5′-GCA CAA GAG GCC CUA GAU UTT-3′; antisense 5′-AAU CUA GGG CCU CUU GUG CTT-3′) for highest knockdown efficiency, and chose siRNA-Pten-c (Figure S4). The negative scrambled control siRNAs lacked significant sequence homology to any gene (sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′; antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′). DCMECs were either transfected with siRNA-Pten-c (Pten siRNA) or negative control using LF2000 according to the manufacturer’s protocol (Invitrogen); untransfected cells were also included as controls. DCMECs were cultured in 6-well plates overnight; for each well to be transfected, 1 µg of siRNA and 2 µL of LF2000 were diluted in 200 µL of OPTI-MEMI medium. The siRNA-LF2000 mixtures were incubated at room temperature for 20 min and then added to well. Cultures were incubated with serum- and antibiotic-free medium at 37°C for 48 h. Optimal transfection conditions were screened in advance (Figure S3).