Fig. 7: Quantitative microscopy of an 8-hour time-course of Bafilomycin A1 (BafA) treatment demonstrates differing cycling behaviour of Golgi proteins.

HeLa cells were left untreated in full growth medium (0 h) or were cultured in the presence of 100 nM BafA for 0.5, 1, 2, 4, 6 or 8 h, before fixation, immunofluorescence labelling and semi-automated widefield imaging. Brightness levels for display of representative images were set uniformly across conditions for each channel. a Representative images of cells labelled with anti-GALNT2 (red). In the merged image, phalloidin-488 labelling of actin (green) and DAPI labelling of the nucleus (blue) are also shown. Scale bar: 10 µm. GALNT2 localization at the Golgi does not change over the 8 h time-course. Note, these are the same cells as shown in panel (c). b Schematic detailing our automated image analysis pipeline for determining the distribution of target proteins between the Golgi (GALNT2 region) and the rest of the cell (phalloidin region). A decrease in the corresponding ratio indicates that a target protein is moving out of the Golgi into other parts of the cell. c, e, g, i, k Representative images of cells labelled with c anti-GLG1, e anti-TM9SF2, g anti-TGOLN2, i anti-GOLIM4, or k anti-SDF4 (all green). In the merged image, anti-GALNT2 labelling of the Golgi (red) and DAPI labelling of the nucleus (blue) are also shown. Scale bars: 10 µm. Note, the target proteins predominantly appear in the Golgi at 0 h, demonstrated by overlap with GALNT2, but their signal in the Golgi decreases as the time-course progresses. d, f, h, j, l Quantification of the ratio of d GLG1, f TM9SF2, h TGOLN2, j GOLIM4, or l SDF4 labelling intensity between the Golgi and the rest of the cell, over the 8 h time-course. Source data are provided as a Source Data file. Each datapoint indicates the normalized log2 ratio for an individual cell (bar indicates median; n ≥ 125 cells per condition examined over two independent experiments). A Kruskal–Wallis test with Dunn’s post-test was performed for comparisons to the 0 h timepoint: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns p > 0.05. See statistics and reproducibility section and Supplementary Table 1 for further details.