Anti rab5

The anti-synj1 (rabbit polyclonal Ab, Novus, RRID: AB_11047653), anti-pTau AT8 and Tau-5 (ThermoFisher, RRID: AB_223647 and 10980631), anti-Rab5 (Santa Cruz Biotechnology, RRID:AB_628191), anti-β actin and tubulin (Santa Cruz Biotechnology, RRID:AB_476697 and 477498), anti-holoAPP MAB348 and 6E10 (Millipore RRID:AB_94882 and 564201), anti-beta-Amyloid (Cell Signaling Technology, RRID: AB_2056585), anti-MAP2 (Abcam, RRID:AB_297885), anti-dynamin clone 41 for (BD bioscience; RRID:AB_3976413), anti-mouse and rabbit HRP (ThermoFisher, RRID:AB_2556542 and 2540618), Texas-Red and Alexa₅₅₅ conjugated anti-mouse and rabbit IgG (ThermoFisher, RRID:AB_10374713, 10983944, 2535987 and 1090271) were purchased. AAV2-containing miR-195, miR-374, scramble controls and miR-195 inhibitors were generated and obtained from ABM Inc. with detailed sequence information available (Am00100, Amm1017200 and Amm3026700). The miRNA extraction kit and qPCR probes for specific miRNAs were purchased from Exiqon Inc. The qPCR probes for actin (Hs1060665_g1), synj1 (Hs00953234_m1 and Mm01210539_m1), gapdh (Mm99999915_g1), RNU6B (NR_002752), 18 s and 45 s (4331182, Mm03928990_g1) were also purchased from ThermoFisher.

Permeabilization was done with 3 % Triton X-100 in PBS followed by 10 % BSA in PBS incubation. Primary anti-Nodal (1:25, rabbit, H-110, Santa Cruz Antibodies), anti-Nestin (1:200, Promega), anti-EEA1 (1:100, Santa Cruz sc5939), anti-Rab5 (1:100, Santa Cruz sc46692), Rab7 (1:100, Santa Cruz Antibodies sc6563) and Rab11 (1:100, Santa Cruz sc9020) were used followed by secondary antibody incubation (1:300, Molecular Probes) plus DAPI nuclear stain (1 μg/1 μl). The slides for Nodal immunostaining were incubated with tyramide (1:100, TSA kit #13, Life Technologies, T-20923) and were mounted with Vectashield (Vector Laboratories). Leica TCS SP5 AOBS confocal microscope was used. The images were handled in Image J. Intensity and co-localization analysis was perfomed by Leica Application Suite (LAS), Leica Microsystems and Pearson’s correlation coefficient (PCC) was used for statistic quantifying colocalization. We analyzed three different fields for each marker, where we performed the co-localization analysis in a mean of three spheroids or 20–30 cells per field. The prevalence of Nodal colocalization with different endocytic markers in each cell line was analyzed by Tukey’s test. For comparison and analysis of Nodal/endocytic markers between two cell lines, we used Sidak’s multiple comparisons test.

HeLa cells, cultured in DMEM (10% FBS 1% Pen/Strep), were plated in 6‐well plates. 24 h later, cells were counted and transduced with vector particles at a particle per cell ratio of 1,000. 24 h later, cells were harvested by extensive trypsin treatment and PBS washing steps to remove any membrane bound vector particles. Cells were counted and 3–5 × 10⁵ cells were used for subcellular fractionation, while 10⁵ cells were analysed by flow cytometry (CytoFlex S platform, Beckman Coulter) to determine transduction efficiency. Subcellular fractionation was performed as previously described (Rossi et al, 2019). Membrane, cytosolic and nuclear fractions were collected. Purity of fractions was confirmed by Western blot using anti‐Rab 5 (1:100, sc46692, Santa Cruz), anti‐Tubulin (1:5,000, T5198, Sigma Aldrich), anti‐Lamin B1 (1:5,000, 16048, Abcam) and anti‐Calreticulin (1:100, PA3‐900, Affinity BioReagents) antibodies. Fractions were subjected to DNA isolation (Qiagen, DNeasy Tissue kit) followed by qPCR analysis (FastStart essential DNA green master reagent, Roche) using CMV promoter‐specific primers on the LightCycler 96 real‐time PCR system (Roche). The specificity of target DNA amplification was confirmed by melting‐curve analysis. All samples were run in technical duplicates.