Fig 2: ARF1-dependent cargo loading into secretory vesicles is PAQR11 dependent.(A) Western blot analysis of whole cell lysate (WCL) and vesicle- and Golgi-fractionated H1299 cell lysates to confirm enrichment of fractionated lysates in organelle-specific markers. ER, endoplasmic reticulum. (B and C) Western blot analysis of FAM3C and PLAU in WCL and Golgi- and vesicle-enriched fractions of siRNA-transfected H1299 cells (B). Protein levels relative to siCTL controls quantified densitometrically (C). (D) FAM3C and PLAU expression constructs. (E) Confocal micrographs of H1299 cells transfected with expression constructs. Vesicles containing ectopic FAM3C and PLAU were detected using fluorescent tags (EGFP-FAM3C) or antibodies against Flag (PLAU). Golgi localization determined on the basis of colocalization with the trans-Golgi marker Golgin-97. Nuclei were counterstained with DAPI (blue). Scale bar, 8 µm. (F) Contrast-adjusted confocal micrographs of H1299 cells cotransfected with Flag-PLAU and indicated siRNAs and stained with anti-Flag antibodies. Arrows indicate Flag-PLAU+ vesicles. Dotted lines indicate cell boundary. Scale bar, 10 µm. Vesicle numbers per cell (dots) were quantified. (G) Schematic illustration of a working model in which p53 loss leads to PAQR11 up-regulation, initiating an ARF1-dependent secretory process that is amplified through an autocrine PLAUR-dependent signaling pathway. Results represent means ± SD values from a single experiment incorporating biological replicate samples (n = 3, unless otherwise indicated) and are representative of at least two independent experiments carried out on separate days. P values, two-tailed Student’s t test for two-group comparisons and one-way ANOVA test for multiple comparisons.

Fig 3: BIG1 mediated-ARF3 activation was essential for LPS-stimulated inflammatory response.a WT and BIG1-/- BMDMs were stimulated with LPS (100 ng/ml) for 30 min, the total, membrane-bounded and cytosolic proteins were measured by Western blot with the indicated antibodies. b Cell lysates from WT BMDMs transfected with Myc-ARF1 or Myc-ARF3 were subjected to pull-down assay, followed by western blot with indicated antibodies. c Cell lysates from WT BMDMs with or without LPS (100 ng/ml) treatment for 30 min were subjected to immunoprecipitation with BIG1 antibody, followed by Western blot with the indicated antibodies. d WT and BIG1-/- BMDMs were treated with LPS (100 ng/ml) for 30 min, the activated ARF3 (ARF3-GTP) was pulled down by GST-GGA and visualized by Western blot. The densitometry of ARF3-GTP was quantified by Quantity One software and the activity of ARF3 in WT and BIG1-/- BMDMs was measured. e WT BMDMs transfected with Si-ARF3 or negative control siRNA were treated with or without LPS (100 ng/ml) for 30 min. Total lysates were subjected to Western blot with indicated antibodies. f WT and BIG1-/- BMDMs were transfected with Si-ARF3 or negative control siRNA for 48 h before treated with LPS for 6 h. The mRNA levels of TNF-a, IL-6, and IL-1ß were measured by RT-qPCR. g, h BIG1-/- BMDMs transfected with active mutant ARF3 (Q71L) or vector were treated with or without LPS (100 ng/ml). Total lysates were subjected to Western blot after treatment for 30 min (g), and the mRNA levels of TNF-a, IL-6, and IL-1ß were measured by RT-qPCR after treatment for 6 h (h). i, THP-1-derived macrophages transfected with negative control siRNA (NC) or si-ARF3 were treated with or without LPS (100 ng/ml) for 30 min. The cell lysates were subjected to immunoblotting with the indicated antibodies. j THP-1-derived macrophages transfected with negative control siRNA or si-ARF3 were treated without or with LPS (100 ng/ml) for 6 h. The expression of TNF-a, IL-6, and IL-1ß mRNA were measured by RT-qPCR. Data show pooled technical replicates from three independent experiments (d, f, h, j). All immunoblot data are representative of three independent experiments with similar results. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test in f and j; one way ANOVA in d, h).

Fig 4: Model depicting proposed PCLX-001 mechanism of action in B cell lymphoma.a Upon BCR activation, first the myristoylated SFK Lyn is recruited to the lipid raft domains of the plasma membrane containing the BCR, dephosphorylated Lyn at Y507 leads to its activation and autophosphorylation at Y396. This leads to the phosphorylation and activation of BTK at Y551 and Y223. Second, myristoylated HGAL is also recruited to the plasma membrane and phosphorylated thereby enhancing BCR signaling by stimulating SYK, BTK and the release of Ca++ ions from the endoplasmic reticulum via the inositol-3-phosphate ion channel receptor (IP3R). Altogether these early signaling events lead to transcription activation by c-Myc, P-ERK, NF?B, and CREB. b The NMT inhibitor PCLX-001 prevents the myristoylation of Lyn-SFK (as well as other SFKs not shown in this model), HGAL and Arf1 thereby impeding the proper membrane targeting and function of these proteins. PCLX-001 treatment impedes calcium homeostasis by reducing the BCR mediated Ca++ release from the ER and increasing basal Ca++ levels in cells in addition to promote the degradation of both myristoylated (Lyn, HGAL, Arf1) and, surprisingly, non-myristoylated proteins (NF?B, P-ERK, c-Myc and CREB), some via the ubiquitin-proteasome pathway thereby further abrogating downstream BCR signaling and increasing ER stress leading to apoptosis and cell death.

Fig 5: Arf1 ablation induces the infiltration and activation of immune cells in Lgr5/Apc mice.a, b Immunofluorescent staining of GFP and CD8a (a) or CD4 (b) in the intestine of the indicated mice. c–e Flow cytometric analysis of gut APCs: DCs (c), inflammatory DCs (d), and macrophages (e) (n = 3 mice each group; **p < 0.01, t-test; repeat two independent experiments). f–m Flow cytometric analysis of the immune cells of LPLs: CD4+ T cells (f), IFN?+CD4+ T cells (g), CD8+ T cells (h), IFN?+CD8+ T cells (i), ?d T cells (j), NK cells (k), Treg cells (l), and IL-13+ CD4+ T cells, (m) (n = 3 or 5). n–p Flow cytometric analysis of the immune cells of IELs: CD4+ T cells (n), CD8aß+ T cells (o), and CD8aa+ T cells (p) (n = 5 mice per group; *p < 0.05, **p < 0.01, t-test; repeat two independent experiments). q–u Relative gene expression of the indicated chemokines, cytokines, and granzymes (n = 5 mice each group, repeat two independent experiments). v Immunofluorescent staining of GFP and pZap70 in the intestine of the indicated mice (n = 5 mice each group; *p < 0.05, **p < 0.01, ***p < 0.001, t-test; repeat two independent experiments). Each experiment replicate two times. Data are shown as the mean ± SEM. Scale bars are as indicated.