Gfp rab7 wt

For transient transfection of hippocampal neurons, 300 ng of plasmid DNA were transfected using 1 μl of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) in 100 μl of the plating medium. Neurons were transfected at DIV 8 and were fixed after two additional days of incubation. Plasmids used were as follows: pEGFP-N1 (Takara Bio, Mountain View, CA, USA), mCherry-Rab5CA (Q79L; Addgene, #35138), mCherry-Rab5DN (S34N; Addgene, #35139), mRFP-Rab5 (Addgene, #14437), GFP-rab7 WT (Addgene, #12605), GFP-rab7 DN (Addgene, #12660), EGFP-Rab7A Q67L (Addgene, #28049), EGFP-Rab4A (Addgene, #49434), EGFP-Rab4AQ67L (Addgene, #49475), EGFP-Rab4AS22N (Addgene, #49476), HA-Rab11-DN (S25N), (Addgene, #101046), HA-Rab11-WT (Addgene, #101047), EGFP-Rab11AQ70L (Addgene, #49553), HA-Rab8a-WT (Addgene, #101048), HA-Rab8a-DN (T22N; Addgene, #101049), and HA-Rab8a-CA (Q67L) (Addgene, #101050). Inhibitors were dissolved in dimethylsulfoxide (DMSO), HGF (Merck KGaA, Darmstadt, Germany) in PBS and added to hippocampal neurons (DIV 9) to final concentrations of 50 ng/ml for HGF, 200 nm for mTOR inhibitor rapamycin (Merck), and 1 μM PHA-665752 (Merck KGaA, Darmstadt, Germany). Analyses were performed 30 min and 24 h (HGF Stimulation) or 24 h (Rapamycin treatment) after the addition of the reagents.

The expression plasmids, GFP-rab7 WT and pcDNA3-CXCL12-sfGFP were acquired from Addgene (#14436 and #98961, respectively). MSCs (5 × 10⁵) were transiently transfected by electroporation with 2 µg of plasmid DNA using an Amaxa Nucleofector 2B with Human Mesenchymal Stem Cell Nucleofector™ Kit (#VVPE-1001, Lonza Biosciences) according to manufacturer protocols. Cells were incubated at 37 °C for 5 min post-transfection and seeded on 6-well plates to be cultured for 24 h prior to be processed for downstream experiments. Under these conditions, approximately 3–5% of the transfected cells ectopically expressed the transgene.