Figure 9. Effect of PIKfyve inhibition... | Rockefeller University Press

Figure 9.

$Effect of PIKfyve inhibition on PFF-induced α-syn aggregation and neuronal death in iNs. (A–C) iNs transiently expressing α-syn-YFP were cultured for 10 days with or without 4 µg/ml PFFs, in the presence or absence of 100 nM Apilimod. PIKfyve inhibition reduced the accumulation of α-synuclein aggregates. Images are representative of two biological replicates. Scale bar: 10 µm. (D–G) iNs were grown for 10 days with or without 4 µg/ml PFFs, in the presence or absence of 100 nM Apilimod or 1 µM Vacuolin. Cells were stained with calcein AM to identify live cells and BOBO-3-iodide to label dead cells prior to imaging. Representative images from three biological replicates are shown. Scale bar: 10 µm. (H–M) Quantitative analysis of PIKfyve inhibition on PFF-induced neuronal death. Data represent imaging fields with values averaged and expressed as mean ± standard deviation. Data are based on at least two biological replicates, with each data point representing observations from 20 to 40 imaging fields, containing 10–25 iPSCs or iNs per field. Statistical significance (P < 0.0001) is indicated by a star; “ns” denotes no statistical difference. (H) Bar graph showing the fraction of live and dead iPSCs over a 10-day period with or without 4 µg/ml PFFs. Apilimod (100 nM) was administered either throughout the entire 10 days or during the last 6 days. Cells were passaged every 3 days at a 3× dilution. Aliquots were plated in 8-well glass-bottom chambers and imaged on days 6 and 10, following incubation with calcein AM and BOBO-3-iodide. (I–K) Bar graphs summarizing the fraction of live and dead iNs not expressing α-syn-YFP or those stably or transiently expressing α-syn-YFP, subjected to the same PFF and Apilimod or 1 µM Vacuolin-1 treatment as described in H. (L) Schematic representation of the experimental design and summary of results for the setup depicted in M. (M) iNs transiently expressing α-syn-YFP were treated with 100 nM Apilimod throughout the entire 10-day period or restricted to the last 2, 4, 6, or 8 days. An intermittent schedule involved alternating 2-day incubation periods with Apilimod, beginning on day 1 of the 10-day period.$

Effect of PIKfyve inhibition on PFF-induced α-syn aggregation and neuronal death in iNs. (A–C) iNs transiently expressing α-syn-YFP were cultured for 10 days with or without 4 µg/ml PFFs, in the presence or absence of 100 nM Apilimod. PIKfyve inhibition reduced the accumulation of α-synuclein aggregates. Images are representative of two biological replicates. Scale bar: 10 µm. (D–G) iNs were grown for 10 days with or without 4 µg/ml PFFs, in the presence or absence of 100 nM Apilimod or 1 µM Vacuolin. Cells were stained with calcein AM to identify live cells and BOBO-3-iodide to label dead cells prior to imaging. Representative images from three biological replicates are shown. Scale bar: 10 µm. (H–M) Quantitative analysis of PIKfyve inhibition on PFF-induced neuronal death. Data represent imaging fields with values averaged and expressed as mean ± standard deviation. Data are based on at least two biological replicates, with each data point representing observations from 20 to 40 imaging fields, containing 10–25 iPSCs or iNs per field. Statistical significance (P < 0.0001) is indicated by a star; “ns” denotes no statistical difference. (H) Bar graph showing the fraction of live and dead iPSCs over a 10-day period with or without 4 µg/ml PFFs. Apilimod (100 nM) was administered either throughout the entire 10 days or during the last 6 days. Cells were passaged every 3 days at a 3× dilution. Aliquots were plated in 8-well glass-bottom chambers and imaged on days 6 and 10, following incubation with calcein AM and BOBO-3-iodide. (I–K) Bar graphs summarizing the fraction of live and dead iNs not expressing α-syn-YFP or those stably or transiently expressing α-syn-YFP, subjected to the same PFF and Apilimod or 1 µM Vacuolin-1 treatment as described in H. (L) Schematic representation of the experimental design and summary of results for the setup depicted in M. (M) iNs transiently expressing α-syn-YFP were treated with 100 nM Apilimod throughout the entire 10-day period or restricted to the last 2, 4, 6, or 8 days. An intermittent schedule involved alternating 2-day incubation periods with Apilimod, beginning on day 1 of the 10-day period.