Fig. 3: IQSEC1 activates ARF5/6 in distinct locations within protrusions.

a Western blot of cells expressing Scr, ARF5 or ARF6 shRNA using anti-ARF5, ARF6 and GAPDH (loading control) antibodies. ARF intensity normalised to Scr quantified. Mean ± s.d., n = 3 independent experiments. p-values, one-way ANOVA. **p ≤ 0.01. b and c Phase images of acini (mVenus positive, yellow) described in (a). Scale bars, 100 μm. Heatmap, area and compactness measurements Z-score-normalised to control. p-values; one-way ANOVA, greyscale values as indicated. n = 3 independent experiments, 5 replicates/condition, 2880–3188 acini/condition in total. Cartoon, acini phenotype representative of each condition. d Western blot of cells co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) or ARF5-mNG and ARF6-RFP (ARF5/6), and Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH (loading control for ARF5) antibodies used. n = 2 independent experiments. e and f Phase images of acini described in (d). Scale bars, 100 μm. Heatmap, area and compactness measurements Z-score-normalised to control. p-values, one-way ANOVA, greyscale values as indicated. n = 2 independent experiments, 4 replicates/condition, 1254–1567 acini/condition in total. g Cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2. Merged and magnified image of spindle cell shown. Arrowheads, colocalization. n = 3 independent experiments. Scale bars, 20 μm. h Acini expressing ARF5-mNG or ARF6-mNG were stained for IQSEC1 (FIRE LUT). Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. Arrows, lack of colocalization. n = 3 independent acini imaged. Scale bars, 5 μm. i Schema, GTPase cycle, site of action of NAV-2729, QS11 and GTP-loaded ARF detection by GGA1-NGAT. j Acini expressing ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections and a single Z-slice is shown. Arrowheads, colocalization in protrusions, arrows, colocalization in cell body. n = 2–5 independent acini imaged. Scale bars, 10 μm. k and l Cells expressing GGA1-NGAT and k ARF5-NG or l ARF6-NG were transfected with Scr or IQSEC1 KD4 shRNA then treated with NAV-2729 or QS11. % overlap of ARF and ARF-GTP probe/cell is shown in box-and-whiskers plot: 10–90 percentile; + mean; dots, outliers; midline, median; boundaries, quartiles. n = 2 independent experiments, 3 replicates/condition with k 541–1287 and l 390–798 cells quantified/condition. p-values, one-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. m Schema, localisation of IQSEC1 and active ARFs in protrusions.