Figure 3.
ER trafficking and interaction with BIP chaperone
(A) Proximity ligation assay for AAV9 intact capsid and BIP protein in iPSC-CMs (upper panel) and iPSC-CFs (lower panel). Scale bar, 10 μm. (B) Quantitative analysis of PLA for BIP-AAV9 as number of spots normalized to number of nuclei. Student’s t test vs. control ∗p < 0.05. (C) Immunofluorescent staining of BIP and protein aggregates (PROTEOSTAT probe) in iPSC-CFs after BIP silencing. Scale bar, 10 μm. Analysis of scAAV-CMV-GFP transduction efficiency: percentage of GFP+ cells and MFI in iPSC-CFs (D and E) and iPSC-CMs (F and G) after silencing of BIP expression. Vectors were added 72 h after siRNA. Multiplicity of infection (MOI) of scAAV9 for iPSC-CFs was increased to 105 from standard 104 for better visualization of the silencing impact. Analysis of scAAV-CMV-GFP transduction efficiency: percentage of GFP+ cells and MFI in iPSC-CFs (H and I) and iPSC-CMs (J and K) in the presence of HA15 assessed by flow cytometry 5 days after exposure to the vectors. Student’s t test vs. appropriate control ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. (L) Immunofluorescent staining of BIP and AAV9 intact capsid, 6 h after exposure to scAAV9-CMV-GFP vectors. Scale bar, 10 μm.