Fig. 2

Ethanol activates the inflammasome pathway in NPCs. NPCs were obtained from iPS cells by epigenetic neural induction and treated with ethanol for 24hr or 7d. a From the top: phase contrast pictures show that the cell density between the different treatments is comparable at day 7. Scale bar: 200 μm. Immunofluorescence analysis by confocal microscopy shows the expression of neural multipotency markers Nestin, Pax6, and Sox2 in NPCs. Scale bar: 50 μm. b Graphs showing the relative percentage of Nestin⁺, Pax6⁺ and Sox2⁺ cells over the total number of DAPI⁺ nuclei. c Fluorescent images of EdU⁺ nuclei after incorporation assay for 24hr at day 7 of ethanol exposure. Scale bar: 50 μm. d Graph showing the relative percentage of EdU⁺ cells over the total number of DAPI⁺ nuclei. e Growth curve of NPC lines #1 and #2 after acute or chronic ethanol exposure. f Immunofluorescence analysis by confocal microscopy shows the expression of the inflammasome markers Casp1 and NLRP3, autophagy marker LC3B, and of the apoptotic marker Casp3 in NPCs with or without ethanol exposure (24hr or 7d). Scale bar: 20 & 50 μm (LC3B). g Graph showing the relative percentage of Casp1⁺ cells over the total number of DAPI⁺ nuclei. h Western Blot and graph showing the relative densitometric analysis of Casp1 (p45 and p10) expression. Quantification of relative protein expression normalized to VCP expression. i Western Blot and graph showing the relative densitometric analysis of NLRP3 expression. Protein quantification was normalized to β-actin. j Graph showing the relative percentage of LC3B puncta per cell. k Graph showing the relative percentage of Casp3⁺ cells over the total number of DAPI⁺ nuclei. The differences among all the values were not statistically significant unless indicated (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). Student’s t-test was utilized for all experiments