Silencer sirna

Silencer siRNA was purchased from Thermo Fisher Scientific against human Rubicon, ATG7, and STX17. Custom Silencer siRNA was ordered from Thermo Fisher Scientific against human LAMP2A (sense: 5′-GGCAGGAGUACUUAUUCUAGU-3′).

C2C12 cells were transfected with an oligonucleotide for follistatin siRNA (Silencer siRNA, 50 μM; Cat# s66250, Thermo Fisher Scientific, Waltham, MA, USA) or Silencer Negative Control #1 siRNA (50 μM; Cat# AM4611, Thermo Fisher Scientific) at day 5 using Endo-Porter (Gene Tools, Philomath, OR, USA), according to the manufacturer’s protocols. The Silencer siRNA is designed for maximum potency and specificity using a highly effective and extensively tested algorithm.

The siRNA oligonucleotides for MTHFR (ID: HSS106762) and MTHFD1 (ID: HSS106759) were prepared using Stealth RNAi™ (Thermo Fisher Scientific, Waltham, MA, USA). siRNAs for GART (ID: s735) and FOLR1 (ID: s5330) were prepared using Silencer^® siRNA (Thermo Fisher Scientific). siRNA for DHFR (ID: SI00299992) was prepared using FlexiTube siRNA (Qiagen, Venlo, The Netherlands). siRNA for TYMS was prepared using MISSION^® siRNA (Sigma-Aldrich). The sense and antisense strand sequences of TYMS siRNA were 5′-CAAUCCGCAUCCAACUAUUTT -3′ and 5′-AAUAGUUGGAUGCGGAUUGTT -3′, respectively. The control siRNA oligonucleotide was Stealth RNAi™ Negative Control Med GC (Thermo Fisher Scientific). Cells were plated into 6-well plates at a density of 2.5 × 10⁴ cells per well, incubated overnight, and then treated with RNAi duplex-Lipofectamine^® RNAiMAX (Thermo Fisher Scientific) complexes (final concentration, 10 nM) for 24 h.

BeWo cells were seeded in 24-well plates at a cell density of 150.000 cells/well and incubated overnight. Afterwards, cells were differentiated with 20 µM forskolin for 48 h. Thereafter, cells were transfected with a predesigned siRNA targeting human SLC2A1 (2.5 pM, Silencer siRNA, ThermoFisher Scientifc) or non-targeting negative control siRNA using Lipofectamine (Lipofectamine™ RNAiMAX Transfection Reagent, ThermoFisher Scientific) as a transfection reagent in 1 ml serum free culture medium for 24 h.

E17 fetal mouse skin was divided under a microscope into the superficial (S) and deep (D) layer of the dermis. Fibroblasts were established from these tissues, and those at passage 5 were used as mesenchymal cells. They were cultured as previously described^{22 (link)} in two-dimensional culture using a regular adherent culture dish (Nunclon Delta,Thermo Fisher Scientific) and in three-dimensional culture using a dish that was made non-adherent by coating it with 1% agarose gel. Next, 10% fetal bovine serum and Dulbecco’s modified Eagle medium with 1% penicillin–streptomycin were used for each culture. Cells were grown at 37 °C and 5% CO₂, and the medium was changed twice a week. Three-week-old cultures were then incubated with Lipofectamine 2000 (11,668–019; Life Technologies, Invitrogen, Germany) and then transfected with Twist2 siRNA (160,520 and 61,236, Silencer™ siRNA; Thermo Fisher Scientific) or non-targeted siRNA (4,390,843, Silencer™ Select Negative Control No. 1 siRNA; Thermo Fisher Scientific) via injection into the culture medium. After 72 h, RNA was collected from the cells using ISOGEN (Nippon Gene), and specific gene knockdown was evaluated via RT-qPCR.

Briefly, 6 × 10⁵ cells were seeded in each well of four-well plates. Transfection was performed using HiPerfect transfection reagents (QIAGEN, France) and 10 ng of miRNA (QIAGEN, France) or 10 nm of Silencer siRNA (Thermo Fisher Scientific, France), according to the manufacturers’ recommendations. For siRNA controls, transfection control (HiPerfect transfection reagent only) and a negative control (silencer negative control 1 siRNA) were used. For miRNA controls, transfection control (HiPerfect transfection reagent only) and an oligonucleotide (miScript inhibitor negative control; QIAGEN, France) were used.

Male C57BL/6 mice were purchased from the Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). Young (15 weeks old) and old (90 weeks old) mice were both used in this study. The complexes were prepared by combining invivofectamine 2.0 (Life Technologies, Carlsbad, CA, USA) and SFRP4 siRNA (152089, 152090, and 152091; Silencer™ siRNA, Thermo Fisher Scientific), according to the manufacturer’s instructions. The SFRP4 siRNA complex, negative siRNA (Silencer™ Negative Control No. 1 siRNA, Thermo Fisher Scientific) complex, or control (PBS) were injected at 4 mg/kg into old mice by intravenous injection weekly (1.5 mg/kg in week 1 and 1 mg/kg in weeks 2 and 3). Experiments were performed with n = 5 for each group. One week later, the skin was collected in whole layers, and tissue specimens were fixed by immersion in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin and Masson’s trichrome staining. Tissues for RNA recovery were later immersed in RNA (Qiagen, Hilden, Germany) and stored at −20° C until use.