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. 2021 Sep 21;10:e68910. doi: 10.7554/eLife.68910

Figure 5. Similarities between Golgi dispersal induced by nicotine and nocodazole.

(A) Comparison of Golgi fragmentation in nocodazole-treated and/or nicotine-treated α4β2R cells. Cells were transfected with St3-GFP (green). Cells were treated (or left untreated) with 10 μM nicotine for 17 hr and further treated for 4 hr with 25 μM nocodazole. Cells were fixed, permeabilized, and immunostained with anti-GM130 antibody (red). Scale bar, 10 μm. Inset scale bar, 5 μm. (B) Quantification of the number of puncta per cell displaying St3/GM130 overlap (top) or St3 only (bottom). Data are shown as mean ± SEM, for St3/GM130 overlap, control cells, 3.1 ± 1.0; nicotine cells, 34.6 ± 9.0; nocodazole cells, 73.3 ± 6.0; nicotine and nocodazole cells, 78.5 ± 9.3 (n = 10 cells per group, con vs nic, *p < 0.035; con vs noc, ***p < 0.00003; con vs nic+ noc, ***p < 0.000003; nic vs noc, p < 0.007; nic vs nic+ noc, p < 0.003) and for St3 only, control cells, 62.6 ± 6.9; nicotine cells, 124.1 ± 8.9; nocodazole cells, 104.5 ± 6.9; nicotine and nocodazole cells, 130 ± 10 (n = 10 cells per group, con vs nic, ***p < 0.00005; con vs noc, **p < 0.004; con vs nic+ noc, ***p < 0.000005). (C) Effect of nicotine on microtubule stability. α4β2R cells were transfected with St3-GFP (green) and the microtubule binding protein, Ensconsin-mCherry (red). Cells were treated with nicotine and/or nocodazole as in A. Scale bar, 10 μm. (D–E) Comparison of the effect of nicotine and nocodazole on primary cortical cultures. Cultures of neurons (DIV 10) were transfected with HA-tagged α4β2R subunits, St3-GFP and Ensconsin-mCherry for 24 hr. Neurons were untreated, treated with 1 μM nicotine or 8 μM nocodazole for 17 hr. Surface α4β2Rs on neurons were labeled with anti-HA antibody (α4β2R-HA; blue). Scale bars, 10 μm. (D) Images of Ensconsin-mCherry (red), St3-GFP (green), and α4β2R-HA (blue) in the somata of untreated (top), nicotine- (middle) and nocodazole- (bottom) treated neurons. (E) Same as in D except for dendrites.

Figure 5.

Figure 5—figure supplement 1. Histograms displaying the size distribution of St3/GM130 (left) and St3-only (right) puncta (8–10 cells per group).

Figure 5—figure supplement 1.

α4β2R cells were treated with 10 μM nicotine for 17 hr, and further treated for 4 hr with 25 μM nocodazole. Parallel sets of cells were left untreated, treated with nicotine alone (17 hr), or nocodazole alone (4 hr).