Fig 2: Panx1 expression remains unchanged. (a) Example of HMs after 4-h incubation in Krebs (sham), TBOA, nicotine + TBOA, or nicotine, labeled with the neuronal marker SMI 32 (left column; green), or Panx1 (middle column; red). Merged images are shown on the right column where DAPI (blue) is used as nuclear marker. (b) Histograms quantifying unchanged levels of Panx1 among the treatments described above. (c) Example of western immunoblotting (top) showing the unchanged expression of Panx1 in brainstems incubated as described above

Fig 3: Expression of P2X7R and Panx1 in INS-1E cells. (a) Representative gel of P2X7 and Panx1 mRNA expression. (b) Representative Western blot of P2X7R and Panx1 expression in INS-1E cells grown in increasing glucose concentrations. Loading control was ß-actin. The images (in a and b) are cropped from original full-length gels and blots that are shown in the supplementary material. (c,d) Western blot quantification of P2X7R and Panx1 (isoform A and D) expression in INS-1E cells grown in increasing glucose concentrations. The ratio of protein (P2X7R or Panx1) to ß-actin were normalized to 2.8 mM glucose. Data represent means ± s.e.m. and n denotes the number of independent experiments and significant differences *p < 0.05 and **p < 0.01 are indicated.

Fig 4: Cholesterol depletion reduces lateral mobility of Panx1 in the membrane. N2a cells transiently expressing mPanx-1-GFP were subjected to treatment with 5 mM methyl-ß-cyclodextrin (MßCD), 5 mM MßCD followed by addition of cholesterol (MßCD + Chol) or left untreated (CTRL). (A–C) Fluorescence images of transfected cells (control and treated with MßCD alone or with cholesterol) before (pre-bleach) and at 0.6 and 60 s postbleach; bleached regions are shown at higher magnification in insets. Bars: 10 µm; inset magnification 3×. (D) FRAP recovery curves for control cells and with MßCD alone or with cholesterol. (E) Histograms of normalized FRAP recovery at 15, 30, and 60 s after photobleach. p values obtained from ANOVA test followed by Dunnett’s test. n = 7–10 per plasma membrane domain, data collected over four independent repeats. ** p < 0.01.

Fig 5: Expression of endogenous Panx1 is largely absent in CRISPR-deleted N2a cells. Representative confocal images obtained from (A) parental, (B) Panx1-deleted, and (C) Panx1-deleted N2a cells expressing human (h) Panx1 construct. Boxed white areas show the Panx1 staining at higher magnification. Scale bars: 50 μm lower magnification, 20 μm in boxed areas.