Fig 1: Effect of rosuvastatin and betulin on neuroserpin aggregation and cholesterol level.(a) Hela-GFP and Hela cells were mixed 1:1, treated with 0.1 μM rosuvastatin or 20 μM betulin for 8days, fixed in 3.7% paraformaldeide and incubated with the primary antibody anti-neuroserpin (Ab32901, Abcam) coniugated PLA probes (1:50), overnight at 4 °C. The presence of aggregates are visualized using Duolink in Situ staining according to the manufacturer’s instructions. Nuclei are stained with DAPI. Images are acquired by Leica SP2 laser scanning confocal microscope. Each red spot represents a neuroserpin aggregate. The number of spots per cell for each condition in the images showed in this panel is quantified as described in the methods section. (b) Untreated or treated cells with 0.1 μM rosuvastatin or 20 μM betulin (8 days) were fixed with 3.7% paraformaldeide and then incubated with TNM-AMCA (1 μM) to stain cholesterol for 1 h at room temperature. Images were acquired by Leica SP2 laser scanning confocal microscope.
Fig 2: Duolink In situ Staining of neuroserpin in HeLa untretaed or treated with SIM or PRA.(a) Hela-GFP and Hela cells were mixed 1:1 and treated with 5 μM SIM or 10 μM PRA up to 8 days, fixed in 3.7% paraformaldeide and incubated with the primary antibody anti-neuroserpin (Ab32901, Abcam) coniugated PLA probes (1:50), overnight at 4 °C. The presence of aggregates are visualized using Duolink in Situ staining according to the manufacturer’s instructions. Nuclei are stained with DAPI. Images are acquired by Leica SP2 laser scanning confocal microscope. Each red spot represents a neuroserpin aggregate. (b) The number of spots per cell for each condition reported in panel a is quantified as described in the methods section.
Fig 3: Aggregation of neuroserpin with statins or botulin.(a) 100 μg protein of HeLa cells or treated with 10 μM PRA or 5 μM SIM or 0.1 μM rosuvastatin or 1 μM rosuvastatin or 20 μM betulin were loaded on 10% native gel and transferred on PVDF membrane. The sheet was incubated with anti-neuroserpin (ab32901, Abcam, 1:500) overnight at 4 °C and then with the secondary antibody anti goat-HRP (1:5000, ECL Blotting reagents (GE Healthcare RPN2109)/SuperSignal West Femto Maximum Sensitivity Substrate, Thermo scientific) for 1 h at room temperature. Ponceau S solution (P7170, Sigma) was used to verify the correct loading of the samples. (b) Effect of statins or betulin treatments on the level of expression of neuroserpin or HMGCR. 10 μg total protein were loaded on 10% polyacrilamide gel, transferred on PVDF and incubated with anti-neuroserpin (ab32901, AbCAM, 1:500) or with anti-HMGCR (1:1000, MAB5374, Millipore) overnight at 4 °C). Vinculin (1:10000, Sigma) was used as housekeeping.
Supplier Page from Abcam for Anti-Neuroserpin antibody