2200 analyzer

To measure cellular acetyl-tubulin levels, cells were seeded, treated, fixed, and permeabilized as described above. Tubastatin was used as a positive control [85 (link)]. After blocking, cells were exposed to mouse monoclonal anti-acetyl-tubulin antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). Average cellular acetyl-tubulin intensity was automatically quantified for each acquired images. Data were standardized over the non-treated control (100%) and expressed as mean ± SD of at least quadruplicates from one assay. At least 1 × 10³ cells were analyzed for each treatment.

To assess if the test compounds induce some level of genotoxicity, cells were seeded as previously described and treated with 10 μM test compounds. Since γ-H2AX signal can decrease over time due to DNA repair, this assay captured SCQ-induced DNA damage after a short treatment period of 4 h. Celastrol was used as a positive control [87 (link)]. After fixation, permeabilization and blocking, cells were exposed to mouse monoclonal anti-phospho-Histone H₂AX antibody (1:1000 in blocking buffer, 15 µL, overnight). After exposure to goat anti-mouse Alexa Fluor 488 secondary antibody (1:10,000, 15 µL, 1 h), cells were stained using DAPI and stored in PBS (Figure S2), images were aquired using an INCell 2200 analyzer (10× magnification) and analyzed using IN Carta image analysis software as described above (GE Healthcare, Rydalmere, NSW, Australia). The number of γ-H₂AX-positive cells was automatically quantified for all acquired images. Percentage γ-H₂AX-positive cells was expressed as mean ± SD of at least quadruplicates from one assay. At least 500 cells were analyzed for each treatment.

Fibroblasts were cultured in chamber slides for 72 hrs after passage and washed with PBS twice, fixed with 4% PFA at room temperature for 15 min, and permeabilized using 0.1% saponin. Slides were blocked with 5% donkey serum and 1% BSA for 30 min, incubated in primary Abs overnight at 4°C followed by incubation in appropriate secondary Abs, mounted in Vectashield solution containing DAPI (Vector Laboratories). Antibodies used for this method were: rabbit polyclonal anti-EEA1 Ab (Abcam), rabbit polyclonal anti-Rab11 Ab (Abcam), rabbit monoclonal anti-Rab7 AB (Cell Signaling), mouse monoclonal anti-CD63/LAMP3 Ab (H5C6; Developmental Studies, Hybridoma Bank), and Alexa fluor secondary donkey anti-rabbit/mouse Abs (Invitrogen). Cells were imaged with a Zeiss 510 META confocal laser-scanning microscope. For quantification of CD63 staining, 6000 cells/well were seeded in 96-well black/clear bottom plates and stained with anti-CD63/LAMP3 Ab, followed by donkey anti-mouse Alexa fluor secondary antibody. Cells were then imaged on the InCell 2200 Analyzer and quantified as described elsewhere [11 (link)].