Neon transfection system 100 l kit

Coronin 1 cloned in pEGFP-N1 served as the wild type control for the mutants. pEGFP-N1 was the vector control while site-directed mutagenesis was carried out to mutate serines 9, 311, 356, 412 to alanine and glutamic acid using primers given in Table S1. RNAi-resistant coronin 1 constructs were generated by mutating the region targeted by RNAi (ACTGGACGAGTAGACAAG toACTGGACGTGTGGACAAG with the mutated residues in italics) to nucleotides present in the same region of human coronin 1 by site directed mutagenesis using primers indicated in Table S1. The RNAi mutant Cor1-EGFP constructs were denoted with an (*) at the end. Transfection was carried out initially using Amaxa Nucleofector kit V (Lonza; program T-20) or the Neon Transfection system, 100 µl kit (Invitrogen) using the program: 1720 V, 25 sec and 1 pulse. Fluorescent cells were sorted using a FACS Aria III (Becton Dickinson) and either used directly for localization studies or expanded for immunoprecipitation, immunoblotting and flow cytometry studies.

Human SGBS cells were kindly supplied by M. Wabitsch (University of Ulm) and cultured as previously described^{13 (link)}.
pCMV plasmid transfections were performed with the Neon Transfection System 100 µl kit (Invitrogen). The electroporation protocol was optimized to pulse voltage 1,300 V, pulse width 20 ms, pulse number 2 and a cell density of 6 × 10⁶ cells ml⁻¹. SGBS cells were transfected with 5 µg of pCMV-AHCY plasmid, for AHCY overexpression or the pCMV-Entry (both oriGene) as a control, per 1 × 10⁶ cells. After electroporation, 500,000 cells per well were seeded in a 12-well format for differentiation assay.

Neon™ Transfection System 100 µL Kit (Invitrogen, Germany) was used to perform the electroporation‐based transfection of MV4‐11 cells. Before electroporation, MV4‐11 cells were cultivated in RPMI media until they reached 70%–80% confluence. On the day of transfection, 5 million cells resuspended in 100 µL of Opti‐MEM media along with 10 µL of either si‐YBX1 (Cat No: 4390824, Thermofisher Scientific, Germany) or si‐scramble (Cat No: sc‐37007, Santa Cruz Biotechnology, Germany) (2 µM‐ final concentration) were electroporated under various pulse conditions (1200 V+, 1350 V+, and 1500 V + 35 ms + 1 pulse) using Neon™ Transfection System (Invitrogen, Germany). After electroporation, the transfected cells were seeded in a 60 mm Petri dish containing 10 mL of RPMI media. To assess the transfection efficiency, plasmid‐encoding GFP was utilized. Suitable pulse condition to downregulate YBX1 expression in MV4‐11 cells was determined using ELISA, western blot and qRT‐PCR. In addition, cell viability was also assessed by using MTT assay (Sigma Aldrich, Germany) and absorbance was measured at 540 nm. Once the right pulse condition was determined, the same experiment was repeated in more Petri dishes to isolate sEVs from MV4‐11 si‐YBX1 and MV4‐11 si‐scramble cells.

HIB1B and 3T3L1 cells were transfected using the Neon Transfection System 100 µL Kit (Invitrogen, Carlsbad, CA, USA) as previously described [53 (link)]. Electroporation parameters were: pulse voltage of 1300 V, pulse width of 20 ms and pulse number 2. For electroporation, gene-specific ON-TARGETplus SMARTpool small interfering (si)RNAs (siCobl, siMyoc and siMkx) and ON-TARGETplus control reagent (Dharmacon, Lafayette, LA, USA) were used at a final concentration of 500 nM and a cell density of 6 × 10⁶ cells/mL.
After electroporation, cells were seeded in 6-well cell culture plates, cultured to confluence and differentiated as described before. The efficient knockdown of gene expression was confirmed on day 0 of adipocyte differentiation using quantitative real-time PCR.

One day prior to transfection, SVF cells were plated at a density of 1400 cells/cm² for optimal growing conditions. The transfection was performed using Neon Transfection System 100 µL Kit (Invitrogen; Thermo Fisher Scientific, Inc.). Before transfection, cells were harvested via trypsination and washed with DPBS.
For PLSCR4 overexpression, 2 µg of PLSCR4 plasmid (PLSCR4 pcDNA3.1+/C-(K)DYK #OHu03299C; GeneScript) or control plasmid (c-Flag pcDNA3; gift from Stephen Smale; Addgene plasmid #20011; http://n2t.net/addgene:20011 (accessed on 26 August 2022) [26 (link)]) was added to the cell pellets, which were resuspended in 110 µL R-buffer for transfection via electroporation in Neon 100 µL tips at 1300 V, 2 pulses, and 20 ms. Afterwards, cells were transferred to prewarmed medium and seeded for functional assays as described below. A change in medium was performed after 24 h.