The chaperone protein Hsc70 facilities cyclin D1 package into EVs. (A) Characterization of APEX-mediated proximity biotinylation of cyclin D1 protein targets by blotting with streptavidin. Cyclin D1–APEX fusion gene was delivered into N2A cells by lentivirus infection. Biotinylated protein was detected by blotting with streptavidin (SA)-HRP. Ponceau S staining (left) of the same membrane served as loading control. (B) Table showing MS analysis of the unique peptides in biotin-phenol together with H₂O₂ (B+H) or without H₂O₂ (B). (C) CoIP analysis of Hsc70 and Hsc90 with cyclin D1 and CDK4 in N2A cells. (D) CoIP of cyclin D1 and Hsc70 in PC12 cells. (E) CoIP of cyclin D1 and Hsc70 in 5 × 10¹⁰ RA-EVs. (F) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells treated with VER-155008 (VER). N2A cells pretreated with RA-containing differentiation medium for 4 d, after which cells were exposed to fresh differentiation medium with or without 5 µM VER for two more days. EVs collected from 6-d differentiation of N2A cells. (G) Immunoblots of cyclin D1, Alix, and CD9 in EVs collected from the differentiated N2A cells transfected with WT Hsc70 (WT) or D10N mutant Hsc70 (D10N; >50% transfection efficiency). WT Hsc70 or D10N mutant Hsc70 were transfected by Lipofectamine 2000 in seven plates of 70%-confluency N2A cells in DMEM medium for 10 h, followed by a change to fresh differentiation medium for 3 d. EVs were collected from both cells. (H) Expression analysis of Pax6, Nestin, and Six3 in differentiated mESCs treated with 2 × 10⁹ EVs from RA-induced N2A cells with (VER-EV) or without (RA-EV) VER. EVs were collected as described in F. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (I) Immunoblots of Hsc70 and actin in control or Hsc70 sgRNA–transfected N2A cells. dCas9 was stably expressed in N2A cells by lentivirus (dCas9), Lentivirus was then used to introduce Hsc70 sgRNA1/2 by transfection of dCas9 cells. (J) Expression analysis of Pax6 and Nestin in differentiated mESCs treated with 2 × 10⁹ EVs from RA-induced N2A dCas9 cells or Hsc70 sgRNA–transfected cells. Values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples.