NUCB1-KO impairs the trafficking of MMP2. (A) Fluorescent images of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP with or without NUCB1-WT, counterstained against NUCB1 (red) and captured after 0, 15, 30, and 45 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (B) Cytoplasmic vesicle counts as described in A are plotted as number of vesicles per cell (n ≥ 90 cells, median ± IQR of two independent experiments; ***, P < 0.001; n.s., not significant). (C) Confocal microscopy images of HeLa or NUCB1-KO cells expressing LyzC-SBP-eGFP and counterstained against NUCB1 (red) after 0, 20, 40, and 60 min of biotin incubation. Arrowheads, cytoplasmic vesicles. Scale bars, 5 µm. (D) Cytoplasmic vesicle counts from C of two independent experiments (n ≥ 42 cells, median ± IQR). (E) Secretion assay of HeLa or NUCB1-KO cells expressing SS-MMP2-SBP-eGFP or LyzC-SBP-EGFP and incubated with biotin for 45 or 60 min, respectively. WCL, whole-cell lysates. [SNs], 10×-concentrated supernatants. (F) Semiquantitative analysis from three independent experiments, one-sample t test. Bars, mean ± SD. (G) GFP-coIP of HeLa or NUCB1-KO cells expressing LyzC-eGFP, with or without NUCB1-WT. GFP-HA, negative control; CN, HeLa control; KO, NUCB1-KO. (H) Semiquantitative analysis of NUCB1 to GFP signal from three independent experiments. Bars, mean ± SD; paired t test.